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ANS124 - 2025 Lab 10: BIOINFORMATICS/Genbank LAB REVISED 3/15 - some text in yellow/red is there just to address a couple of boo boos that were in the previous version Please note. At end of lab we will dedicate ~10 minutes to the teaching evaluation. We will set aside time in Discolab for you to do these so no need to do them before or after lab. Thanks in advance from Russ and TA's for completing these, as they do matter/count. LOCATIONS March 11 from 2-4 - 2216 TLC March 12 from 1-3 - 75 Hutch March 12 from 3-5 – 2020 Esau March 13 from 2-4 - 2216 TLC What you have to do: After lab this week (week10), upload your Assignment which are questions in this document. You are welcome to stay in computer lab and get any help to complete those at end of DiscoLab. Due by the start of final exam on Friday 21 March (Advice is to knock them out straight after DiscoLab - it will be much easier!!!) Please use these questions and this manual as a study guide for the final. Provide answers to the 10 questions (in green) in this lab manual - answering them as we go, as they will help complete the assignment and review material. Ignore the previous reference to questions A-E. Step 1: Re-visit your gel from last week, it was dried down onto filter paper after it was stained. Some of these we did last week already (so its a recap). First – find the top of the gel (where the wells were). Second – recognize that it might have been “swapped” as it was prepared for drying (ie the ladder you loaded in lane 1 might suddenly be in lane 12, and all other lanes similarly out of order, ie read right to left, not left to right) See if you can see the “ladder”. Determine size of the other “rungs” relative to this one maybe. Band “migration” (distance traveled) is inversely proportional to the molecule’s size. Ie. large molecules at top of gel. The band “intensity” (darkness) is proportional to the number of molecules of that particular size. Hence, a dark band means there is more of that protein compared to a faint band. Compare some of our “pure” proteins (xanthine oxidase, alpha lactalbumin, beta lactoglobulin) to the different milk preparations you loaded. Can you see certain bands in the milk – and are they in the samples that “make sense that they should have those proteins”?? See if you can speculate what the molecular size of some of these proteins is? PART 1: BIOINFORMATICS - What is bioinformatics? Bioinformatics is the analysis and modeling of biological information using computers and statistical techniques. The primary goal of bioinformatics is to increase our understanding of biological processes. Many computer tools exist for this purpose, ranging from databases to store molecular information (sequences of genes and proteins), programs to analyze sequence similarity, predict genes, find patterns, predict 3D structures, etc. With bioinformatics, you can use "dry-lab" techniques to help design experiments, analyze data and deduce function. By contrast, last week you did a "wet-lab" biochemical analysis. Why bioinformatics in ANS124? You separated out milk proteins on a polyacrylamide gel. This resulted in a gel containing several bands, and you may already have an idea about what those bands might represent. In this lab, you will gather information about milk proteins and the genes that encode them, using your computer, that will back up your attempts to identify the bands on your gel. This lab will give you some hints and tips that might help you to find information about any protein or gene that you’re interested in the future. First, choose a protein: We will be studying the following milk proteins: αs1-casein, αs2-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, butyrophilin. Q1: Can you recall in what fraction of the milk each of these proteins exists? Part A: Finding a nucleotide sequence The Genbank Nucleotide Database is a collection of sequences. The database is produced in an international collaboration with EMBL (Europe) and the DNA Database of Japan (DDBJ). Each of the three groups collects a portion of the total sequence data reported worldwide, and all new and updated database entries are exchanged between the groups on a daily basis. Genbank can be accessed via the website of the NCBI (National Center for Biotechnology Information). Step 1. Go to the NCBI website (www.ncbi.nlm.nih.gov), and select “nucleotide” from the pull down menu in the search box. Type in the name of your protein, including the species ‘cow’ (use its Latin name!) – you will get a list of many hits. Have a look at some of them and try to work out how they are either similar or different. Genbank contains sequences in many shapes and sizes: genomic DNA fragments (ie bits of chromosomes), transcripts (mRNA) with “coding regions”, allelic variants, etc. Every sequence entry has a unique accession number, an entry date, and author information. Information about the sequence itself can be found under ‘FEATURES’ such as source (organism, type of sequence), coding sequence (CDS), etc. Step 2. From the results, select the RefSeq tab to narrow down your results. Genbank is a collection of publicly available sequences: anyone can submit a sequence to Genbank. It’s basically the author’s responsibility to provide correct, useful information about the sequence. Reference Sequence (RefSeq) provides an integrated, non-redundant, curated set of sequences, including genomic DNA and transcripts, and is created by NCBI. Step 3. Pick an mRNA sequence with a complete coding sequence of your favorite gene. You can select it from the Genbank or the RefSeq results – just make sure that the sequence contains the complete CDS and not a partial CDS (read on for a description of CDS). Q1b What is “Coding Sequence”? Q1c Genes are made of regions (genomic DNA) called? Q1d How does the gene sequence differ from the mRNA sequence? Q2. For the bovine protein you are working with in discolab, what is the accession number, the length of the submitted sequence, and the length of the CDS? Q2a) How many exons does your sequence have? (Information about exons/introns is not always available for all sequences). If part of the nucleotide sequence encodes a protein, a coding sequence (CDS) (complete or partial) will be given, as will the translated protein sequence (remember the letter symbols for the different amino acids?). Q3. How long is the bovine protein that is encoded by your nucleotide sequence (the one from Q2), i.e. how many amino acids? What is the first amino acid? Q4. Remember that a triplet of nucleotides codes for one amino acid (called what?). Does the length of the bovine protein sequence make sense with regard to the length of the coding nucleotide sequence? Step 4. In the description of the coding sequence (CDS) there should be a protein ID. For your bovine protein/CDS write this down, we will need it later on. Step 5. At the right hand side of the screen, you will find a box with the header ‘All links from this record’. Click on the link ‘Gene’. This will bring you to the record for your sequence in Entrez Gene, a searchable database of genes from species for which the (partial) genome sequence is available. Q5. On what bovine chromosome is the gene coding for your favorite bovine protein located? What are the neighboring genes? (dont worry about this question we didnt have time) Hint: have a look at the section ‘Genomic context’. Part B: Comparing the nucleotide sequence of one species to another species Step 6. At the top of the page you can choose between different formats. At the moment you are (probably) looking at the GenBank format. Click on ‘FASTA’ (this is a style of sequence presentation), this will give you the nucleotide sequence in FASTA format. Copy the sequence. Step 7. If you go back to your GenBank format, you will see ‘Run BLAST’ under ‘Analyze this sequence’. Click on ‘Run BLAST’. This will bring you to the BLAST entry page. In the ‘Enter Query Sequence’ you will find the accession code of your selected sequence. Alternatively, go to the BLAST homepage (www.blast.ncbi.nlm.nih.gov) and paste your saved FASTA sequence or accession number here. Make sure the selected database is nucleotide collection (nr/nt) (this is the default). Hit the BLAST button at the bottom of the page. BLAST (Basic Local Alignment Search Tool) is a program that searches nucleotide databases for sequences that match your entry sequence. Think about the probability that one sequence is the same as another in the body - how would you work this out? You can compare sequences for one species to another species, or look for sequences that match your unknown sequence. Step 8. Scroll down to the long list of matching sequences. Probably the first hit is the sequence you have entered. From this list, look for the homologs (similar entries) of your sequence in sheep, goat and human (what are the Latin names for these animals?) Q6. Record the accession number in the following space of the homologous sequence in sheep, goat and human. Try to find a sequence with a complete CDS.Note, if for example human doesnt show on the list (because of too many ruminants!) then you might go back to the BLAST page and in the "organism" box type "homo sapiens" (which tells it just to compare to human) and then at bottom select "somewhat similar sequence, BLASTN" (what this does is it tells the search to only look at human sequences, and tells the algorith to not be soo picky!!). See what happens - you cant break it!!.. Step 9. If you click on the accession number of the homologous sequence, you will get the respective GenBank entry. If you click on the number in the ‘Max score’ column, you will jump to an alignment of your entry sequence and the matching one. Q7. What is the identity percentage of your entry sequence with the sheep, goat and human sequences? Please write the percentages of each here. Dont stress too much about the exact percent, we are just asking you to see what is close or not.. some trends might appear. And if you have Beta-lactolobulin, maybe you wont find the human... why not? Part C: Finding a protein in the SwissProt protein database The exercises above were all about nucleotide sequences (mRNA and DNA). Now we’re going to see what information is available for our favorite protein in the Swissprot protein database. “UniProtKB/Swiss-Prot is a curated protein sequence database which strives to provide a high level of annotation (such as the description of the function of a protein, its domains structure, post-translational modifications, variants, etc.), a minimal level of redundancy and high level of integration with other databases”. Step 10: Open the Swissprot website( www.uniprot.org ) and enter the protein_id (see Step 4) of your protein (‘Search’ at top right). This will result in 1 or more hits. Click on the accession number of your protein. Failing this - you can always just type in your "Bos taurus your milk protein" into the search box - and then try and select a "Gold Star" option - these are more likely to have been checked, validated, and annotated. Swissprot gives you tons of information: general info (accession number, name and origin of the protein, etc.), description of function, cross references, variants, etc. Q8. Most proteins are produced as a precursor. A so-called signal-peptide is cleaved off later, yielding the mature protein. Using the PTM window, how many amino acids is the signal peptide of your protein? What do you think is the function of this signal peptide? Q8a. Does your protein have any post-translational modifications? (note you may have to scroll down on this screen as its a box in a box) Q9. What is the mass of your protein (in daltons = Da)? Q10. Now you know more about milk proteins and its sizes, do the bands on the SDS-PAGE gel from last week make sense? E.g. are the bands the expected size? If no, could you think of an explanation?