Yeast Spotting Predictions and BLAST Analysis
(15 POINTS) Due Next Lab
In this assignment you will complete a table of predictions Media:Hypothesis for yeast rev 2010.doc for the growth phenotypes of the various yeast strains and write a brief analysis that includes your findings from a BLAST (Basic Local Alignment Search Tool) search of the Sec18 and Sec61 protein sequences using the Saccharomyces Genome Database (SGD) and the National Center for Biotechnology Information (NCBI) Database. In these databases you will look for information about the function of the protein products of SEC18 and SEC61 and gain knowledge about their importance and evolutionary relationships by searching for homologs in other species. In writing the summary of your analyses, you may also want to consult some of the references on the yeast secretory pathway that have been posted to your lab conference.
When you feel that you understand the different roles of Sec18 and Sec61 in the secretory pathway,
- Complete the hypothesis table provided, making predictions about which yeast transformants will grow on which types of media and at which temperatures.
- Write a brief analysis that includes:
- A summary of what you have learned about the steps in the secretory pathway that are mediated by the Sec18 and Sec61 proteins with primary literature citations - NOT SGD
- An explanation of the rationale for your growth phenotype predictions - you SHOULD NOT explain each square - you should be able to look at this sheet in columns (SD-U media) and rows (SD-U-H media) to tell your reader what you are controlling for!
- A brief discussion of what you learned from your BLAST search for homologs (human and others) about the extent that these 2 proteins have been conserved and what that might indicate about the importance of the proteins.
You may have time to begin this assignment during lab today. Predicting the growth phenotypes will probably be a challenging exercise and should be started first so that you can benefit from working with your partner or other classmates and from consultation with your instructor. Make sure you understand the rationale behind each of the predictions you submit. Your written summary must be completed individually.
Directions for using the NCBI and SGD Databases
Finding basic information about protein function in SGD
Go to the Saccharomyces Genome Database (SGD) website, http://www.yeastgenome.org/. This database is a portal through which you can access all sorts of information about yeast genes and proteins. In turn, look up both SEC18 and SEC61 using the search box at the top right of the page (the search is case insensitive). You will probably be able to identify the basic functions of the two gene products relatively quickly, but take some time to explore the various links to familiarize yourself with the various types of information that can be obtained from SGD. In particular, scroll down to the "Protein Information" section of the page and click on the right-hand link for "All protein evidence and references." This will open another page for the protein product of the gene that contains a lot of useful information, such as the molecular weight, isoelectric point, predicted sequence features, and full amino acid sequence. Take a look at this information before proceeding. Once you have discovered the functions of the two yeast proteins, start on your yeast growth predictions before beginning the search described below for homologs of the gene products.
Finding information about potential homologs through a BLAST search
To search for proteins in other species that have sequence similarity to Sec18 and Sec61, you will perform a BLAST search. Scroll down the page for each protein to the section entitled "Analyze Sequence." Under the subheader, "S288C vs. other species," click on the link for "BLASTP at NCBI." Note that S288C is the particular strain of S. cerevisiae that was sequenced in the yeast genome project and that NCBI is the National Center for Biotechnology Information. This will open the BLAST page of the NCBI website (http://www.ncbi.nlm.nih.gov) and will automatically paste in the NCBI protein sequence identifier (in the format “NP_XXXXXX.X), where the Xs are numbers) into the search window. Use the default database “nr” and the other default commands. The NCBI database can allow you to compare your protein sequence of interest to other sequences from a vast range of organisms. If you try searching the entire database, you will find that the top hits are all proteins from other yeast or fungi. To narrow the results to a more manageable list and find matches from a more diverse group of organisms, specify all or some subset of the following organisms in the "Choose Search Set" window: Schizosaccharomyce pombe (fission yeast), Caenorhabditis elegans (roundworm), Drosophila melanogaster (fruit fly), Danio rerio (zebrafish), Mus musculus (mouse), and Homo sapiens (humans!). Note that the "+" button by the Organism line will allow you to add additional organisms to the list. After you have specified the search set, click the “BLAST” button, and wait for the results. The page will initially show any conserved domains identified in the protein; you may have to wait several minutes for the complete search to finish and for the homologs to be displayed.
Which of the organisms you selected has a protein with the greatest homology to your yeast query protein? Do all of the organisms have some protein that is relatively similar to the yeast query protein?
Identify the human protein that is most similar to each yeast protein. Note that there may be duplicate entries for the same protein match if there are multiple accession numbers for it in the database. Scroll down to the "Alignments" section below the "Descriptions" list and locate the alignment for the top human "hit." Click on the Locus/Accession Number for that protein. (This will be the descriptor that starts with 2-3 uppercase letters—it will be above the GENE ID.) Scroll down to the bottom of the locus page, where you will find the protein sequence. Find out how many amino acids are in the complete protein. Jot this down along with the protein's GenBank accession number, and then return to the previous page. (When your browser asks you if you want to send a form again, click "Send.") Examine the alignment between the yeast protein and the top human "hit." Does the alignment extend throughout the entire lengths of the two proteins? Record the % sequence identity (IDENTITIES) and the % sequence similarity (POSITIVES). Try to discover the function of the human homologs. (Note: if there are duplicate entries for a match, they may contain more or less functional information, so don't look at just one.)
Briefly summarize what you have learned about Sec18, Sec61, and their homologs in a written homework assignment (1-2 paragraphs). You will also find this information to be useful when you write the lab report at the end of this series.
|| At or Above Standard
|| Below Standard
|| Points Earned
| Prediction Table
|| 5.5-7.5 pts. Accurately predicted growth vs. no growth pattern in reporter strains carrying RSB204 and in control strains transformed with RSB203 or YEP24 based on 25°C as permissive temp. (secretory pathway is indistinguishable in SEC+ vs. sec mutant strains), 30°C (temp. where mutant phenotype is observed), & 37°C temp (where secretory pathway is assumed to be completely blocked).
|| 0-5 0.5-5 pts. Did not accurately predict yeast growth based on temperature sensitivity parameters given, selective media conditions, reporter gene constructs, and/or control constructs & conditions.
-1 pt for each mistake in predicting growth pattern in strain carrying reporter gene (ss-HIS4) on SD-U-H.
-0.5 for mistake predicting growth in control strain (those transformed with RSB203 or YEP24 or on SD-U media)
|| 5.5-7.5 Included a clear, accurate, and literature supported discussion of the function of Sec61 & Sec18; clearly explained reporter; explicitly & correctly related protein function to growth prediction in the reporter strains. Tied BLAST analysis results & identification of closest human homologs into discussion of the importance of yeast Sec18 & Sec61 in secretory pathway function. Cited reference sources properly in the body of the paper as well as in the reference list.
-0.5-1 pts. Missing, incorrect, or unsupported information about the function of Sec18 and Sec61.
-1-3pts. Missing a clear & explicit explanation of how the ss-His4p reporter allows discrimination between where Sec61 & Sec18 act in the secretory pathway.
0.5-1pts. Omitted closest human homolog for either or both Sec18 &/or Sec61 or identified the homolog(s) incorrectly.
1-2 pts. Did not discuss the significance of the BLAST analysis findings.
-0.5 pt. Did not cite sources in proper format in the body of the paper and/or did not include a properly formatted reference list at the end of the discussion.