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. Use the “Quick Search” feature to look up SEC18 and SEC61 (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. Once you have found the function 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. Click on VIEW next to “Protein Info” in the drop down menu called “Protein Info & Structure” on the right side of the SGD main page for the gene of interest. This link opens a homepage 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. Then, scroll down on the Protein Info page to where it says “EXTERNAL LINKS;” click on the “BLASTP (NCBI)” link under the “Homologs” sub-heading. This will open the BLAST page of the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov) and will automatically paste the NCBI protein sequence identifier (in the format “NP_XXXXXX.X), where the Xs are numbers) into the search window. You have probably noticed that SGD has its own BLAST feature (one of the options at the top of the page), but that one only compares an input sequence to the sequences of other yeast proteins. Because we are interested in potential homologs in other organisms, you will perform your BLAST search using the more comprehensive NCBI database, which contains protein sequences from a vast range of organisms. Use the default database “nr” and the other default commands. Click “BLAST!” and follow the directions for viewing the results. You may have to wait several minutes for your search to finish.
To organize the results, click the TAXONOMY REPORTS link (~ one-third of the way down the page), then try out the different ways you can display the data. Look carefully at the human homologs by clicking on the link called “hits” next to Homo sapiens in the LINEAGE REPORT. Click on the LOCUS number (the locus number or accession number usually starts with 2-3 uppercase letters) of the top human “hit”. Find out how many amino acids are in this protein, jot down its locus number, and then close the locus window.
To determine which human protein is most similar to your yeast protein over the whole protein sequence, return to the BLAST results page. Scroll down to find the accession number of the most promising human protein from your Lineage Report results (top hit). NOTE: If you have previously clicked on its locus number, that protein may appear as a different color than the others which could help you find it more quickly. (The homologs are arranged in order of sequence similarity, so the human ones are unlikely to be near the top.)
Once you have found the human protein that exhibits the greatest sequence similarity with your yeast protein, click on number to the right of the locus and protein name in the column called SCORE BITS (DO NOT click again on the locus number!). The SCORE BITS link should take you to the section in the BLAST results containing the alignment of the yeast protein (QUERY) with the hit, the human protein or SUBJECT. LENGTH refers to the number of amino acids in the protein. Look for consensus over the whole protein. Since you know the total number of amino acids in the protein, you can look at the end of the alignment to see how far significant consensus extends.
Record the name of the closest human homologs for both yeast Sec18 and to Sec61. At the top of the alignment section you can find and record the % sequence identity (IDENTITY) and the % sequence similarity (POSITIVES).
Use the taxonomy report to find out the range of organisms that have similarity to the yeast proteins. Try to find the function of the human homologs using other references. Briefly summarize this information for this homework assignment; you may also find the information to be useful when you write the lab report at the end of this series.
- Please note that, for some inexplicable reason, the BLAST program does not always seem to allow you to view the alignment between yeast Sec61 and the top human "hit" when you click on the "SCORE BITS." (This does work for Sec18, however.) To get around this wierd glitch, if it happens to you, you can make your own alignment of the yeast and human proteins using the alignment tool found at: http://www.expasy.ch/tools/sim-prot.html. You can copy the yeast protein sequence from the "Protein Info" link on the Sec61 Saccharomyces Genome Database page and copy the human sequence from the page you get to when you click on the LOCUS number for the top human hit.
|| 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 Sec61p & Sec18p; 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 yeast Sec18p & Sec61p’s importance in the secretory pathway. 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 Sec18p and Sec61p.
-1-3pts. Missing a clear & explicit explanation of how the ss-His4p reporter allows discrimination between where Sec61p & Sec18p act in the secretory pathway.
0.5-1pts. Omitted closest human homolog for either or both Sec18p &/or Sec61p 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.