Two Different and Correct Answers
These answers are provided exactly as the students submitted them.
First Example where student chose one path with good data to support the conclusion.
I first performed an BLASTn with the above sequence. There was 100% identity to part of the S. cerevisiae chromosome XII cosmid 8300. There was 99% identity to the PBR1 gene of S. cerevisiae. There was one gap with this sequence. The S. cerevisiae PBR1 gene for sensitivity to papulacandin B is 5631 bp long, so again, this 660 bp fragment was only a small portion of the gene. Base pairs 1-398 of the query also corresponded with a 100% identity to the Saccharomyces cerevisiae 1,3-beta-D-glucan synthase subunit (FKS1) gene, which is the same length as the PBR1 gene. These appear to be the same gene. In fact, when I performed a BLAST2 search with the two sequences, there was a 99% identity. Please see printout.
Before I realized that both genes were the same length, I had thought that perhaps there was some alternative splicing going on with the sequence, but when I inserted the query sequence into ORFfinder, there was only one significant ORF. Which of course now I know corresponds to BOTH genes because they are the same thing.
When I performed a PubMed search of PBR1, I found four hits, but one in particular caught my interest: “Papulacandin B resistance in budding and fission yeasts: isolation and characterization of a gene involved in (1,3)beta-D-glucan synthesis in Saccharomyces cerevisiae” by Castro C, Ribas JC, Valdivieso MH, Varona R, del Rey F, Duran A. A free copy of this paper was located at the Journal of Bacteriology online. In the paper, the investigators characterized the PBR1 gene in S. cerevisiae. According to the paper, PBR1 is identical to the FKS1 gene, which is part of the 1,3-beta-D-glucan synthase complex. (This complex is responsible for the biosynthesis of a major structural component of the yeast cell wall).
It is also interesting to note that in the BLAST results, some human BACs appeared (very small pieces of BACs) and a few other small pieces of other orthologs. However, with these sequences, the E-values are larger, making them not as biologically relevant. It could be just a coincidence that these sequences showed up at all.
Thus, this particular sequence does not appear that this sequence has any major orthologs. The sequence is part of a larger sequence that produces a particular protein that may or may not be in other organisms. It does have a conserved domain (as evidenced when I inserted the amino acid sequence of the PBR1 gene into the conserved domain database). The conserved domain occurs between amino acids 807 and 1632 and corresponds to glucan synthase. According to the conserved domain site, a glucan synthase catalyzes the formation of beta-1,3-glucan polymer, which again is a major component of the fungal cell wall. (Note that the conserved domain is not part of the query sequence; it is merely part of the protein sequence that the query sequence leads to).
Second Example where student chose a different path with
good data to support the conclusion.
I began by taking this unknown sequence to the BLASTn database. This search
led to a number of hits. Three of these hits appeared to me to be realistic
hits because their E-values were 0 while all other hits had E-values of 0.89
or greater. These three hits were all for yeast. The three red bars were the
yeast hits.
One hit was for a segment of chromosome 12, the next was for the yeast PBR1 gene on chromosome 12, and the last one was for FKS1 on chromosome 12. This information I received by clicking on the NCBI links from the BLASTn results page. In order to determine why these three hits were slightly different I searched PubMed for PBR1. This gave me one paper about the anti-anti-fungal properties of PBR1, but no sequence data. I then checked to see if PBR1 is conserved in humans at GeneCards and the Human Genome Browser and received no hits for this gene. I figured the next best place to go to learn about yeast genomics would be SGD. At SGD I performed another Blastn, this time only of the yeast genome. This blast found a 100% match on chromosome 12 in the ORF YLR343W. I next did a FASTA search at SGD and once again found a 100% match to ORF YLFR343W. I looked at the ORF map of this region of chromosome 12 and was surprised by what I saw.
This map shows that our mystery sequence is in ORF YLR343W, the adjacent ORF to FKS1. Since the original BLASTn data led me to believe that the sequence was from FKS1 (aka PBR1) I retrieved the full sequence for FKS1 and YLR343W. It is in fact the case that our sequence is from YLR343W and not from FKS1. Unfortunatly there is little to nothing known about this ORF. I decided to take the amino acid sequence provided by SGD for our nucleotide sequence and perform a Blastp search. This proved to be circular as the main protein I found was a hypothetical one based on ORF YLR343W. I next did a Conserved Domain Blast with the protein and found that it most closely matched a GAS1 (glycolipid anchored surface protein) domain.
I searched NCBI-protein for YLR343W and found a reference to a probably transmembrane protein of the GAS1 family, thus confirming the above diagram’s assumption. There is also a reference to it being a probable glycoprotein involved in signaling.
