Guide Sheet for tics Lab 1 - 4

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<p>Adopted (with minor modifications) from: www.nslc.wustl.edu/elgin/Bio3055/manual.pdf www.nslc.wustl.edu/elgin/genomics/Bio3055/3055/projects/PAH/PAH_manual.pdf</p> <p>______________________________________________________________________________</p> <p>SCHEDULEBioinformatics Lab 1: Bioinformatics Lab 2: (Guide sheet 1)</p> <p>ACTIVITYTutorial on bioinformatics tools (COX-2 or PTGS2) Start mini-project/exploration on a disease related to PAH (explore Gene, ExPASy, and ClustalW) Answers to Tutorial Sheet Questions Due Answers to Reading Assignment Due Swiss-Pdb Viewer/DeepView Structure Problem Set due Answers to Guide Sheet 1 Questions Due OMIM and KEGG Answers to Guide Sheet 2 Questions Due Answers to Guide Sheet 3 Questions Due Report for Bioinformatics Lab 1-4 Due</p> <p>Bioinformatics Lab 3: (Guide sheet 2) Bioinformatics Lab 4: (Guide sheet 3) One week after Bioinformatics Lab4</p> <p>Note: There is a glossary of terms available in the course website through LMS (if it is not available on the computer desktop). ______________________________________________________________________________</p> <p>BIOINFORMATICS LAB 1: Tutorial to Basic Bioinformatics: COX-2 (PTGS2)This tutorial involves web-based bioinformatics exercise. The tutorial is based on the enzyme cyclooxygenase-2 (COX-2), also known as prostaglandin synthase-2 (PTGS2). The bioinformatics tools from the NCBI (National Center for Biotechnology Information) website will be used. NCBI is a division of the National Institute of Health (NIH), USA. These tools include Gene, GenBank, RefSeq, and PubMed. Gene = database of genes with a brief summary, the common gene symbol, information about the gene function, and links to websites, articles, and sequence information for that gene</p> <p>GenBank = historical database of gene sequences, which means it contains every sequence that was published, even if the same sequence was published more than once. Therefore, GenBank is considered a redundant database. RefSeq = database of sequences that is edited by NCBI and is NON-redundant, meaning that it contains what NCBI determines is the strongest sequence data for each gene. ClustalW, which is a multiple sequence alignment program will also be introduced. ClustalW allows the entry of a series of gene or protein sequences that one believes to be similar and may be evolutionarily related. These sequences are usually obtained by performing a BLAST search. ClustalW then aligns the sequences, so that the lowest number of gaps is introduced and the highest numbers of similar residues are aligned with each other. About COX-2 (PTGS2) This enzyme is important in prostaglandin synthesis. Prostaglandins have a wide range of roles in our body from aiding in digestion to propagating pain and inflammation. Aspirin is a general inhibitor of prostaglandin synthesis and therefore, helps reduce pain. However, aspirin also inhibits the synthesis of prostaglandins that aid in digestion. Therefore, aspirin is a poor choice for pain and inflammation management for those with ulcers or other digestion problems. Recent advances in targeting specific prostaglandin-synthesizing enzymes have lead to the development of Celebrex, which is marketed as an arthritis therapy. Celebrex is a potent and specific inhibitor of COX-2. Celebrex is considered specific because it doesnt inhibit COX-1, which is involved in synthesizing prostaglandins that aid in digestion. This is a remarkable accomplishment given the great similarity between COX-1 and COX-2. This achievement has paved the way for developing new therapies that bind more specifically to their target and therefore have fewer side effects. Understanding the enzyme structures of COX-1 and COX-2 helped researchers develop a drug that would only bind and inhibit COX-2.</p> <p>PART A: Background on Cox-1 (PTGS1) and Cox-2 (PTGS2)step1. Go to the NCBI homepage at http://www.ncbi.nlm.nih.gov step2. Select Gene from the database pulldown menu. Type PTGS in the search box, then click Go. step3. Scan the results for the Homo sapiens entries. There should be one called PTGS1 and one called PTGS2. step4. Select each entry by clicking on its name, then read the paragraph under the Summary section for each entry, and provide answers to the questions below.</p> <p>Q1.</p> <p>PTGS1 and PTGS2 are isozymes. Isozymes catalyze the same reaction, but are separate genes. What types of reactions do PTGS enzymes catalyze? What biochemical pathway(s) are these enzymes involved? How is the expression of PTGS1 and PTGS2 different? Which isozyme would you want to inhibit to stop inflammation? The drug Celebrex selectively inhibits PTGS2 while aspirin and other non-steroidal antiinflammatory drugs (NSAIDs) inhibit both PTGS1 and PTGS2 in the same way. Why do you think researchers wanted to discover a selective inhibitor to PTGS2? Describe how studying 3-D structures of PTGS1 and PTGS2 could help researchers design a drug that binds to PTGS1, but not to PTGS2. (Note: Q4 &amp; Q5 may require an internet search, but be very brief in your answer).</p> <p>Q2. Q3. Q4.</p> <p>Q5.</p> <p>PART B: Getting Sequence Information and Viewing Database Entries NCBI Genestep1. Go back to the Gene entry for Homo sapiens PTGS2. step2. What is the gene name? ______________________ step3. What is the GeneID number? _______________________ step4. Where in the human genome is this gene located? ________________________ step5. What is the RefSeq accession number for the mRNA sequence of Homo sapiens prostaglandin-endoperoxide synthase 2?__________________. step6. Open the entry, then choose FASTA from the pull-down menu. Copy the sequence (including the title line designated by the &gt; symbol) and paste it into a word document. step7. Select the Replace tool under the EDIT menu. In the find box, type ^p to find all paragraph marks. Dont type anything into the replace box. Then click Replace All. This will eliminate all the paragraph marks in the document. If you still see white spaces in the sequence, use the same procedure, but type ^w in the find box to represent white spaces. step8. You now should add back a paragraph mark after the title line (that starts with &gt;) and before the sequence starts. Save the file as Cox2rna.doc in your desktop folder (create one).</p> <p>step 9. What is the RefSeq accession number for the Homo sapiens PTGS2 protein sequence?_________________. Open the entry. Follow the steps given above to save the sequence in FASTA format as a Word document called PTGS2prot.doc file on your desktop. step10. Start a new browser, and go to the Expasy website (Swiss-Prot Entry) and search for the Swiss-Prot entry for PTGS2.(Hint: use the gene name to search and be sure to select the HUMAN protein from the search results).</p> <p>step11. Write at least three alternate names for this protein. ____________________________ step12. Where in the cell is this protein located? _________________________________ step13. What types of drugs target this protein? _______________________________ step14. What amino acid is acetylated by aspirin (amino acid type and number)? _____________ step15. What His residue is in the active site? ____________________ Sequence Manipulation step16. Go to the Sequence Manipulation Suite (http://bioinformatics.org/sms/). step17. Click on Translate under DNA Analysis heading from the menu. step18. Clear the data entry box by hitting Clear. step19. Copy the mRNA sequence from your Word file and Paste it into the data entry box on the Sequence Manipulation website. step20. Select Reading Frame 3 and direct from the pull-down menus, then click Submit. step21. When the Output window opens with your results, copy and paste the sequence into a Word document and save it as, translate.doc on your desktop folder. step22. Compare this sequence in the translate.doc file with the sequence in the Cox2rna.doc. What are the first residues that are the same in the sequences? Do the sequences look like they are the same? ______________________ step23. Open a doc. file on your desktop, SIXclustalW.doc. (or alternatively cox2sixseq.pdf). Using the Text select tool, select all the text and copy or go under the Edit menu and Select All. This file contains six FASTA formatted sequences of PTGS2 from different organisms. The top sequence is the human PTGS2 protein sequence you have been working with. step24. Go to the ClustalW website and enter (by using copy and paste) all of the FASTA formatted sequences into the data entry box. Select input for the Output order so the human sequences will stay at the top in the alignment. Press Run.</p> <p>step25. Copy the alignment and paste it into a Word document. To make this file readable, do the following things: a) Go to Page Set-up under File and change the page orientation to landscape. b) Select all text and change to Courier font, size 10. Courier is the best font for alignments because all the letters are the same width. c) Save this file to the desktop as ClustalW.doc step26. Review the alignment. What symbols are used for positions in the alignment that contain: identical, _________________ highly homologous, ________________ homologous, ________________ and non-homologous residues ________________ Are the residue numbers mentioned in steps 13 and 14 conserved? Why would you expect them to be conserved? _________________________________</p> <p>BIOINFORMATICS LAB 2: Working with Primary Protein Structure InformationIn this lab session a protein from the SwissProt database will be used. One can search for the protein of interest by using the gene name given in Gene. A protein entry in the SwissProt contains some of the same information that is found in Gene, as well as information about the protein sequence, structure, and function that is summarized in a short, easy-to-read format. GUIDE SHEET 1 (below) will provide instructions and guidelines for the internet-based mini project you will be doing today. The ultimate goal for todays lab is to create a multiple sequence alignment for the protein of interest using ClustalW. This alignment software helps to identify the protein mutation, to observe regions of high sequence conservation, and to map secondary structure predictions. The protein mutation is important to identify since it is the basis for your mini project exploration in the bioinformatics component of this lab course and is important for understanding the link between the protein and the disease that will be studied. The regions of high sequence conservation are important because they often correspond to regions in the protein that are important to the proteins function.</p> <p>GUIDE SHEET 1PART 1 OBTAINING THE BASICS: Getting sequence information and viewing the SwissProt and GenBank entries for the protein of interest. Instructions and Guidelines: Follow this guide sheet and answer the questions that accompany this guide as you work through each section. Be sure to refer to hints whenever there are given. Translating the patients cDNA step1. Obtain the mutant cDNA sequence, PAHmutseq.pdf from the course website through LMS (if the sequence is not available on the desktop). Open the file and copy the sequence. step2. Go to the Sequence Manipulation Site (http://bioinformatics.org/sms/). step3. In the menu to the left, Click on Translate found under the heading DNA analysis. step4. Clear the search box, then paste your patients cDNA sequence into the search box. Choose a reading frame from the pull-down menu. Click Submit. step5. You should be able to find the sequence of your protein by finding the first methionine (M), then continuing until you see the first * which is a stop codon. Copy the protein</p> <p>sequence in that region, starting with the first M and paste it into a word document. Save the document in your folder on the desktop. Now you have saved the file of the mutant protein sequence. NCBI Gene step6. Using Gene on the NCBI website, find the entry for the protein you are studying by searching with the protein name. Be sure to select the Homo sapiens protein from the list of results. step7. Open the entry for the RefSeq protein sequence and save the sequence in FASTA format to your desktop folder. Name this file WTprotein.doc so that you will know it is the un-mutated protein sequence. Swiss-Prot Entry step8. Go to the ExPASy website and search for the SwissProt entry for your protein using either the protein name or the gene name. Be sure to select the human protein from the list of results. Make sure the information in the entry is the same as you saw in the Gene entry. If your protein is an enzyme, the EC number is a good way to double-check. You may want to record the SwissProt entry number in case you want to find this entry again. PART 2: BLAST SEQUENCE: FINDING HOMOLOGOUS PROTEINS Protein-protein BLAST step9. Perform a BLAST search using the RefSeq un-mutated protein sequence. First, select PSI-PHI BLAST. Then paste the FASTA formatted protein sequence in the search box. Select the nrprotein database. Click BLAST to begin. On the next page that appears, select Format. You may need to wait a few minutes before the results page opens. After obtaining the results, choose 5 sequences from various positions in the results. The goal is to choose a variety of sequences that differ in evolutionary distance from the human protein. Be sure not to choose any sequences that are human, since they are the same as your search sequence. For each of the 5 sequences, click on the sequence name to view the GenBank entry for the sequence. Then view the sequence in FASTA format. Copy and paste all the FASTA formatted sequences into the same Word file and save it to your desktop folder. At the beginning of this file, add your mutant protein sequence, also in FASTA format. step10. This Word file will be used to create the multiple sequence alignment, so the formatting is very important. The format for this file should be like the example used in the tutorial for COX-2. Review the entry for FASTA format in the Glossary. You should end up with a Word file that contains the 5 sequences from the BLAST search plus the unmutated human protein sequence and your mutant sequence for a total of 7 sequences. Each sequence should be in FASTA format and contain a title line (starting</p> <p>with &gt;, then text, then a return). Shorten the text to contain JUST the species information so it will fit in the alignment (next step). For example, you should erase the gi line and add in something simpler like rat, cat, etc. Your mutant sequence should read &gt;mutant. At the end of each title, be sure to press return to separate it from the rest of the sequence. PART 3 MULTIPLE SEQUENCE ALIGNMENT step11. Go to the ClustalW website and enter (by using copy and paste) all your FASTA formatted sequence into the data entry box. The default parameters should be OK, except for the output order. a) Select input for the Output order b) Press run step12. Save the alignment by copying and pasting the alignment into a Word document. At first the...</p>

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