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| Bioinformatics: A Tutorial for Beginners |
Copyright 2003, Gale
Rhodes, adapted with permission of the author.
IntroductionThis tutorial allows you to explore opsins -- the proteins that catch light for our eyes -- and the genes that code for opsins. But the real subject of this exercise is bioinformatics -- the use of computers to search for, explore, and use information about genes, nucleic acids, and proteins. While learning about the human opsins, you will use some of today's most powerful bioinformatics tools. You can follow up this tutorial with a study of opsins from other organisms, or by exploring any class of biomolecules that interest you. I assume that you are conversant with biochemistry and molecular biology. If you see unfamiliar terms pertaining to the genes, mRNAs, and proteins used as examples here, break out your biochemistry text, head for the index, and review, review, review. For more information about each database or tool, go to its home page and read, read read. Cast of CharactersI. The Databases (and their acronyms!)
II. The Tools
Here We GoOur subject is human opsins, those proteins, found in the cells of your retina, that catch light and begin the process of vision. We will proceed by asking questions about opsins and opsin genes, and then using bioinformatics to answer them. When I provide a web address, I'll also make it a link -- just click it to go to the site. Then make it a bookmark so you can find it again. Where are the opsin genes in the human genome?Point your browser to http://www.ncbi.nlm.nih.gov/mapview/. Read the instructions. Note that you can look at a genome by clicking on the NAME of the species, not the B beside it. The species name takes you to a viewer for the genome of that organism. The B takes you to a BLAST search tool (later). Click Homo sapiens (human). You see a diagram of the human chromosomes, and a search box at the top. Enter "opsin" in the box next to Search for. Click Find. You see the diagram again, with red marks at your "hits", the locations of genes whose entries contain "opsin" as a whole or partial word. Below the diagram is a list of the indicated genes. Among them are the rhodopsin gene (RHO), and three cone pigments, short-, medium-, and long-wavelength sensitive opsins (for blue, green, and red light detection). Four hits look like visual pigments, which probably does not surprise you. To the left of each entry is the chromosome number, allowing you to tell which red mark corresponds to each entry. Note that two opsins are on the X chromosome, one of the sex-determining chromosomes. You can pursue multiple hits on the same chromosome with the all matches link for that chromosome. Click all matches next to X. You see a very complicated display (don't sweat -- we're going to use only a part of this now). On the left is a diagram of the X chromosome, with red marks at the positions of the gene(s) you've followed to this page -- in our case, the two opsins, medium- and long-wave, which are located near the bottom tip of the X chromosome. To the right are various representations of the X chromosome, with listings of annotated areas. The two opsin genes are highlighted in pink. If you pass your cursor over this page without clicking, you will find that some symbols provide brief information, most about regions that are not yet characterized well enough to have a full entry. As you can see, there is a tremendous amount of information on this page, with links to much more. If you want full information about the meanings of abbreviations and symbols on this page, as well as the kinds of information linked to the page, you can use Map Viewer Help at the top of the page. You will find abundant information about the Map Viewer, explanations of all symbols and links, and even tutorials about how to ask and answer all kinds of questions about the genome. For now, note the information provided for the first of the two highlighted opsin genes, OPN1LW (this is called the gene symbol). You see that this is the long-wavelength-sensitive (red) opsin, and that it's a gene involved in color blindness (a sex-linked trait -- no surprise). What do scientists know about the opsins?Click OPN1LW. You have entered LocusLink, which is a sort of highway interchange with routing to all sorts of information about this gene. Scan down the page. Some of the information is very plain and understandable, while some is very cryptic. One of the most accessible links is to OMIM (for Online Mendeliam Inheritance in Man), a catalog of human genes and genetic disorders. Despite the name, the database includes genes of women, too. Click the little orange rectangle labeled OMIM, at the top of the page. This OMIM entry tells you about this gene and colorblindness, a genetic disorder associated with mutations in this gene. Read as much as your interest dictates. Follow links to other information. For more information about OMIM itself, click the OMIM logo at the top of the page. Once you've satisfied your appetite, return to the LocusLink page (use the Back button of your browser or your browser's history list -- if you're lost, click HERE). Click the red PUB rectangle at the top of the page. You have entered PubMed, a free database of scientific literature, to a list of articles directly associated with this gene locus. By clicking on the authors of each article, you can see abstracts of the article. If you are on a university campus where there is online access to specific journals, you might also see links to full articles. PubMed is your entry point to a wide variety of scientfic literature in the life sciences. On the left side of any PubMed page, you will find links to a description of the database, help, and tutorials on searching. Read the abstract of the article by Nathans and co-workers before returning to LocusLink. NB to GR: Add some guided searches in PubMed. What is the nucleotide sequence of this gene?Remember that we are looking at the gene for the red-sensitive opsin in humna vision, and it's located near the bottom tip of the X chromosome. Scroll down to NCBI Reference Sequences (RefSeq). You see that mRNA (messenger RNA) and protein sequences are available, along with a GenBank sequence. Click the entry number beside mRNA. This is a typical GenBank nucleotide file, and a lot of it is hard to read, but a few things are clear. First note, under references, a citations to the publication of this sequence in the scientific literature. To see an abstract of the article in which this gene was described, click the PubMed link below the reference. As you see, you've been here before. There are many ways to move from one database to another, which is both a blessing and a curse. You have to keep your eyes open for useful links, and when you find a path that you think you might use again, make a note of it and bookmark the web pages. It is frustrating to know there's an easier way to do something, and not remember how you did it. NB to GR: point back to this abstract when you get the phylogenetic tree. Scroll to the bottom of this long page. The last thing is the sequence of this messenger RNA. You are seeing the actual list of As, Ts, Gs, and Cs that make up the message for synthesis of this opsin. But wait! You know that RNA contains no T. In most nucleotide databases, U from RNA is represented as T, to make for easy comparison of DNA and RNA sequences. This sequence information is not in the form that is most useful for searching in databases, say, searching for related genes. Let's display this entry in a form more useful for searching. At the top of the page, beside the Display button, pull down the menu that says default (we are looking at the default entry display), and select FASTA (note that several other display options are available). Then click the Display button. You see one descriptive or "comment" line that begins with ">", followed by the nucleotide sequence. This little file is just what you need to search nucleotide databases for similar sequences. Let's keep it for future use. Click and drag on the web page to select everything from the ">" through the last nucleotide. Be careful not to select anything else. From your browser's Edit menu, select Copy to make a copy of this information on your clipboard, for pasting elsewhere. Now start your favorite word processor, make a new document, and paste. The FASTA comment and sequence should appear. Select all of the text and change the font to Courier or Monaco -- these "typewriter" fonts make it easy to align letters into columns, because all letter are the same width. Save this file, choosing text or plain text as the file type. Call it mrnared.txt. Save it to a convenient location for the files you'll be making later. Click your browser's Back button until you return to LocusLink. What is the amino-acid sequence of this gene?Click the entry number beside Protein. Things look a lot like before, but this is a protein entry, containing the amino-acid sequence in one-letter abbreviations. Just as with the mRNA entry, turn this into a FASTA display, and copy it into a new word-processor document. Save it in text format as protred.txt. Return to LocusLink. What does the neighborhood of this gene look like?Click the entry number beside GenBank. Display the entry in default format. This entry shows the sequence of the specific DNA clone that contains the opsin gene, along with information about how this clone was produced. This entry thus shows the gene in the slightly larger context of the cloned fragment in which this gene was found. This sequence would allow you to see flanking regions around the gene, and perhaps to design PCR primers for making useful quantities of the nucleotide sequence so you could express this gene in a cloning vector. From this page, you could also find neighboring sequences if you wanted to look farther afield. As before, display this entry in FASTA format, and save it as a word processor text document entitled GBred.txt. What proteins in humans are similar to the red opsin?Now return to the NCBI Map Viewer. We're going to search the human genome for sequences similar to that of the red opsin. Click the B next to Homo sapiens (human). This is the NCBI's BLAST search tool. BLAST is a widely used program for finding sequences similar to a "query" sequence that you're interest in. Pick these options from the various menus:
Next, copy the FASTA data from your file protred.txt to your clipboard, and paste it into the BLAST search box, above which it says, "Enter an accession..." Check to be sure that the first character in the box is the ">" at the beginning of the FASTA data. Then click Begin Search. The next page is for formatting your search results. Just click that enthusiastic Format! button. When your results are ready, the results of BLAST page appears. Look down the page to the graphical display, a box containing lots of colored lines. Each line represents a hit from your blast search. If you pass your mouse cursor over a red line, the narrow box just above the box gives a brief description of the hit. You'll find that the first hit is your red opsin. That's encouraging, because the best match should be to the query sequence itself, and you got this sequence from that gene entry. The second hit is the green opsin -- remember that the PubMed entry reported that the red and green pigments are the most similar. The third and fourth hits are the blue opsin and the rod-cell pigment rhodopsin. Other hits have lower numbers of matching residues, and are color coded according to a score of matches. If you click on any of the colored lines, you'll skip down to more information about that hit, and you can see how much similarity each one has to the red opsin, your original query sequence. As you go down the list, each succeeding sequence has less in common with red opsin. Each sequence is shown in comparison with red opsin in what is called a pairwise sequence alignment. Later, you'll make multiple sequence alignments from which you can discern relationships among genes. See what you can figure out about what the scores mean. Identities are residues that are identical in the hit and the query (red opsin), when the twoo are optimally aligned.. Positives are residues that are very similar to each other (see residue number 1 in the blue opsin -- it's threonine in red opsin, and the very similar serine in the blue). Gaps are sometimes introduced into a hit to improve its alignment with the query. The more identities and positives, and the fewer gaps, the higher the score. Note that blue opsin and rhodopsin are only about 45% identical to the red opsin. Other proteins, which are apparently not visual pigments, have even lower scores. Now let's take a look at where all these hits are in the human genome. Where are all the genes for these other proteins?Click the Genome View button near just below the introductory information at the top of this result page. You have come full circle. You are back that the human chromosome diagram, and all the hits of your search, in the colors that signify their BLAST scores, are located for you on the diagram. Notice that there are about 100 proteins (discovered so far, that is) that have 40% or more positives in alignment with red opsin. The opsins are members of the very large family of G protein-coupled receptors, key players in signal transduction. How are the opsin genes related to each other?Answering this question requires making a multiple sequence alignment and then using it to make a phylogenetic tree. For these tasks, we move to another database where it's a little easier to gather a bunch of sequences into a single FASTA file. Point your browser to http://us.expasy.org. ExPASY is mirrored at several locations including the following: http://www.expasy.org, http://ca.expasy.org. If one does not work or responds slow, try a different one. You see the home page of ExPASy, the Expert Protein Analysis System. As I said earlier, ExPASy is a complete protein tool box. With ExPASy, you can do almost any imaginable analysis or comparison of protein sequences and structures. Click Swiss-Prot and TrEMBL under Databases. Read the introduction to these databases. They are high quality protein sequence databases with abundant annotation, minimal redundancy, and many connections to other databases. Click Advanced search in Swiss-Prot and TrEMBL. With advance searching, you can limit your search to specific genes and organisms, and you can search on descriptive information in the entries Set up a search for human opsins, as follows:
Click Submit. The page Swiss-Prot description is your search result page. Look over the results. On 9/8/2003, this search gave 14 hits. The rod pigment rhodopsin (OPSD), along with the three cone pigments (OPSB, OPSG, OPSR). There is also a "visual pigment-like receptor peropsin", OPSX. Sound mysterious. Let's find out more about it, and in the process, see a typical Swiss-Prot entry. Click on the gene name, OPSX. You see the NiceProt View of Swiss-Prot: O14718. Persue this entry and try to find out just what this rhodopsin-like protein is thought to do. Under Comments, you'll learn that it's found in the retina (the RPE or retinal pigment epithelium), and that it may detect light, or perhaps monitors levels of retinoids, the general class of compounds that are the actual light absorbers in opsins. Also under Comments - Similarity, you see, as mentioned earlier, that this protein is a member of the large family of G protein-coupled receptors. If you click "G protein-coupled receptors" under the Keywords, you find a list of all purported 7-transmembrane receptor proteins in SwissProt. The human genome alone contains 350 of them! See if you can verify this statement, without counting. Now back up to the NiceProt view. Under References click the journal citation, "Proc. Natl. Acad. Sci. U.S.A. 94:9893-9898(1997). From the resulting page, you can read a full article in the Journal of the National Academy of Sciences (PNAS) about this protein. Like many journals, PNAS puts full articles online just 6 to 12 months after publication. Looking further down the page, you find cross-references to the protein or its gene in other databases, predicted structural features of the protein, and last, the sequence. Note also, at the bottom of the page, links to a number of ExPASy tools listed for further analysis of this sequence. Try some of them. For example, I just learned in about ten seconds from Compute pI/MW that the isoelectric pH (or pI) of this protein is 8.78. And I learned in no time at all from ScanProSite that the sequence contains signatures indicating that the protein is probably a G protein-coupled receptor (no surprise, but comforting) and that it has a retinal binding site. ProSite is a tool for finding signatures of function in new sequences.When you finish playing with these powerful tools, return to your SwissProt search results by use of the back button of your browser. If you're lost, go back to ExPASy and do the search again. Now let's compare the sequences with each other. We'll use the program ClustalW to make a multiple sequence alignment. Scroll down the result page and check the boxes at the left of these entries
At the top of the page, at Send selected sequences to, select Clustal W (multiple alignment) from the menu, and click Submit. ClustalW has been implemented at many web sites. This one, at EMBnet.org, automatically receives the FASTA files from the selected entries, allows you to make some settings of the alignment criteria, and then does the alignment. We will just accept the default alignment settings. First, scroll in the Input Sequences box and verify that it contains five FASTA files, one right after the other. To make them easier to identify in subsequent outputs, edit the name of each FASTA comment line (begins with ">") as follows:
In all cases, be sure to leave the ">" in the first line of each FASTA entry. To save some work in case something goes wrong, select the edited contents of the Input Sequences box, copy it, and paste it onto an empty word-processor page, and save the file in text format. Name it Opsins.txt. Click Run ClustalW. The resulting page is called ClustalW query receipt, and it contains links to several output files. Click clustalw (aln). You see the typical ClustalW alignment file, showing our five protein sequences aligned to maximize identical and similar residues. Below each line of five sequences are symbols to show the extent of similarity among the sequences. An asterisk (*) means that the same residue is always (that is, for all of these sequences) found at that location; for example, the first asterisk marks a location where only N (asparagine) is found. Colon (:) means that all residues at this location are very similar; for example, the first colon is where only F (phenylaline), I (isoleucine), and L (leucine) -- residues with large, nonpolar sidechains -- occur. Period (.) means somewhat similar residues; for example, at the first period, serine, threonine, and glutamine occur -- all polar, but varied in size. If there is no mark then the residues at that location display no predominant common properties. Once more, as a safety measure, copy this alignment to your clipboard, and paste it onto an empty word-processor page. Then save the file in text format. Name it OpsMSA.txt. Remember that it is still on your clipboard, for pasting at our next stop. This multiple sequence alignment is one type of input you can use to make a phylogenetic tree. What does the family tree of human opsins look like?Point your browser to http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html This is one home of the program Phylip, One of the most rigorous tools for constructing phylogentic trees from aligned sequences. Under Proteins, next to protdist, click "advanced form." You are about to run protdist, a program that computes the "distance" of sequences from each other. These so-called distance matrices will be used by Phylip to construct your tree. Enter your email into the top box. In the alignment file box, paste your mutiple sequence alignment from ClustalW. Click "Bootstrap Options" and make these settings:
At the top of the page, click "Run protdist". protdist constructs distance matrices by a process called "bootstrapping". Bootstrapping is a bias-reducing procedure in which protdist builds an alignment of pseudosequences by picking residue positions at random and stringing the residues at those positions together until the sequence is the same length as the original ClustalW alignment. From this pseudosequence alignment, protdist determines the relative number of sequence difference among the five proteins, as determined from a random sampling of their sequences. The result of the process is a called distance matrix, and you will see it soon. This process is repeated, 100 times in our case, to make 100 distance matrices. The tree we will ultimately produce represents a consensus of the 100 matrices. There may be a delay of a few minutes before the result pagee appears. If the server is busy, you may be informed that results are being sent by email. If so, check you email in two or three minutes. You will receive five messages, the first one simply containing a link to your result page. Click the URL, or paste it into your browser and press <return> to open the page. On the Phylip: protdist page that results, click outfile to see the output from protdist. The file contains 100 matrices containing numbers that represent the relative number of differences among the five sequences. Each matrix has the sequence names in the first column, and you should imagine that these sequence names are also the headings for the remaining columns. The number at the intersection of the row Blue and the column with the imaginary heading Peropsin gives the relative magnitude of the sequence differences between the blue opsin and peropsin. The matrices have zeros on the diagonal because each pseudosequence is identical to itself. Click the Back button of your browser to return to the Phylip: protdist page. On the first pull-down menu of the Phylip: protdist page, pick "neighbor." Read the menu carefully: don't pick "weighbor". Click "Run the selected program on outfile" to run Phylip with the output file of matrices you just examined. You are running a procedure called "neighbor joining" to construct an evolutionary tree. On the Phylip: neighbor page that appears next, beside "Distance method?" Make sure "Neighbor-joining" is selected. Click "Bootstrap options" and make these settings:
Scroll down to "Other options". This entry area gives you the optin of designating an outgroup for the root of your tree. An outgroup is the sequence you think is most distant from the others, possibly the commn ancestor of all. We don't know that in this case, so leave the default of 1. At the top of the page, click "Run neighbor". The resulting files are outfile.consense -- your tree, in a text file, and outtree.consense -- your tree in a format used by tree-printing programs. Click on outfile.consense to see the tree. Scroll down to the bottom of this file to see the consensus tree. This tree is "unrooted", meaning that we do not know the ancestor of all these sequences. We learn from this tree which sequences are most alike and which are most different. We also learn how often the connections of this tree were made the same way in the 100 trees made from those 100 difference matrices. The numbers on the branches indicate the number of times that partition of the species into the two sets separated by that branch occurred among the 100 trees. For example, the separation of Red and Green from the other three, indicating that Red and Green are more similar to each other than to the other three, occurred in all 100 trees. The separation of Blue and Peropsin from the other three occurred in only 82 of the 100 trees. In the other 18 trees, Rhodopsin and Peropsin were separated from the other three. (Can you extract this information from this file?) In the tree branching shown, the majority rules, and the results of 18 of the trees are discarded. You can save this file by selecting all and pasting it into a word-processor document. Call it outfile_consense.txt. Return to the Phylip: neighbor page and click on outtree.consense. This is your tree in Newick format, which is widely used by tree-printing programs like Phylodendron. Let's use this program to give us a tree in attractive graphics, rather than text. Point your browser to www.es.embnet.org/Doc/phylodendron/treeprint-form.html. Paste the contents of your outtree.consense file into the Tree Data box. Select Phenogram from among the Tree Styles. From the menu at Extra Options, Output, select GIF format for your output file. Give your tree a title, such as "Human Visual Opsins and Opsin-Like Proteins". Finally, click Submit. Your GIF-format tree appears in your browser window. To keep it, chose Save As ... from the File menu. Call the file OpsinTree.gif. My tree looks like this:  What is the structure of an opsin?By now, I'm particularly curious about peropsin, but it's not likely that the structure of a recently discovered protein of unknown function has been determined. But it is likely that all opsins are similar in structure, so let's see is we can find an opsin in the database for macromolecular structures, the Protein Data Bank (PDB). It will give us an idea of what kind of thing an opsin is. In fact, the PDB does not contain molecular structures at all. It is better to say that it contains models of macromolecules. These models are interpretations of data from one of the two main methods of macromolecular structure determination: x-ray crystallography and NMR spectroscopy. When researchers determine the structure of a macromolecule, they deposit a file containing the three-dimensional coordinates of all the atoms in the model. This coordinate file -- along with an online molecular graphics tool (like **) or a computer graphics program like Deep View -- are all that you need to see and study the molecule on your computer. Next we will retrieve a model from the PDB and view it with an online graphics tool. We'll also visit the home of a topnotch computer graphics program that you can download FREE and use on your home computer. Point your browser to http://www.rcsb.org/pdb/. The PDB home page contains a simple search box under Search the Archive. You can search for models using simple keywords or PDB ID codes. An PDB code has four characters, like 1CYO. How would you ever know a model by its code? When a new structure is published, the authors usually give the PDB code in the last reference of the bibiography. With that code, you can go straight to the model you want to see. But more often, your question, like ours is more general. For such cases, PDB also provides forms for more sophisticated searches. For now, let's just see if any opsin models are availalble. Type "opsin" into the search box and click Find a structure. As of 9/8/2003, this search returns 95 models (on 3/30/2003, it returned 88 models), and you can see from the first one that our search is too broad. Among other things, we're finding netropsin, an antitumor drug. There's also bacteriorhodopsin, and the last time I looked, bacteria had no eyes, so this is not likely to be a visual pigment. Looking over the first two pages of hits, I see one promising sign: some entries for bovine rhodopsin. Some of the hits appear to be fragments of this molecule. So let's use a more precise search tool to see if other bovine rhodopsin models are available. Return to PDB home. In the Search the Archive box, click SearchFields. We have gone from the simplest to the most sophisticated search tool. SearchFields is a customizable form that allows many search criteria. The criteria names are links to the definitions of the criteria, providing information on the contents of PDB files and the criteria that will look in specific parts of the files. At the bottom of the form are criteria you can add to the form. Then you can bookmark a form and always find it with the criteria you want. Now let's get serious and see if there are PDB models that are similar to human rhodopsin. Scroll down to the list of criteria you can add to the form. Check to add these criteria: FASTA search, Ligand and prosthetic groups, and Source. Click New Form. It looks like you have come back to the same page, but now the new search criteria are available. You can now search with a FASTA sequence, you can limit the search to models contains specific nonprotein ligands (like retinal, the prosthetic group of visual pigments), and you can specify the source organism from which the macromolecule is obtained. Find your FASTA sequence of human rhodopsin and paste it into the FASTA Search box. To limit your search to models containing the visual prosthetic group, type "retinal" into the Ligand and Prosthetic groups box. Click Search. This search may take a few minutes. The tool is looking for sequence homology among more than 20,000 entries in the PDB. On 9/8/2003, I got only 5 hits for this search. The first one had PDB code 1LN6, and was listed as a model of bovine rhodopsin. If your search produces other hits, find 1LN6 among them. Beside FASTA result, you see the number 8.8e-155. This is an alignment score meaning that the probability that this entry and the human rhodopsin sequence are similar just by chance is 8.8 x 10-155; not bloody likely, in other words. Click alignment. In a new browser window, you see the alignment between the human rhodopsin sequence and that of 1LN6. After alignment, they are over 90% identical. If two proteins are more than about 40% identical, they are almost certain to be practically identical in structure. So this model will show us what human rhodopsin looks like. Close the alignment window to reveal again the search results. Things to come in the future: -- go the visualization page and use QuickPDB to see the structure -- then download and examine with Deep View (link to tutorial), noting covalent link to retinal -- then examine peropsin: does it have a retinal binding site? use PROSITE within Deep View -- then then make a homology model of peropsin using SwissMODEL -- that should do it. |
