How to use Biology Workbench to find the amino acid sequences of your organisms.

1. 
   Type into the window “ hemoglobin ” (or some other protein) in the box and using the pull down to the right- choose “ show all hits .” See note below on looking at sequence names.
   
2. In the colored windows below (these are various databases one could work with) check the box labeled - “ SWISSPROT .”
   
   
3. Go back up and click – “ Search .”
   

Note: PIR , GENBANK and PDBFINDER may also be use for the initial searches as well. ( PIR1 contains validated sequences whereas others may contain partial or tentative sequences.) GENBANK might be useful to limit searches to a particular order of organisms, such as primates. Limitations of genbank include listing fragmentary portions of genes, which can be confusing to beginning users of the BW tool. PDBFINDER has the 3-D structures for the molecules, if that is desired.

A new page with another window appears with all the retrieved sequences (scroll down to the text box window).

Scroll through the list of results and look at the organisms that have had that particular protein analyzed and submitted to the database. Highlight those in which you are interested. By using [ctrl] and clicking on an organism you can select several organisms to be compared.

Note: It can be hard to tell what some of the organism results show when using show all hits. If you check show 10 results per page, a checkbox list shows up – uncheck those you do not wish to use from a page that has any you need then import the ones you wish to use from that page to your workbench. You will need to use the back page button on the browser (to return to the last searched page) and proceed to the next 10 hits page. Continue this process until you have all the ones you need. If there are none you desire then just go to the next page and ignore un-checking the choices. This is sometimes slower but provides more information at a glance.

Search Strategies and other notes:



1. Input a particular amino acid like myoglobin into BW and select view all sequences. If more than 100 show up you may wish to use a Boolean operator (AND, OR, NOT) to begin to limit the number of “hits” that you obtain.
   
   
2. Input a group of organisms such as whale or canidae and see what comes up.
   
   
3. You will need at least 3-4 different organisms showing up in a particular molecular analysis to be useful in generating phylogenic fragments to develop a composite tree. Make sure the same protein is used in clustered alignments. As an example, avoid mixing alpha and beta strands in these analyses.
   
   
4. There have been some problems in using sequences listed as “tentative” in the search results. Explorations involving these may provide bizarre results, but it may be an interesting sidelight to the activity to try to explain these results. Also, results labeled FRAGMENT may not be as useful unless the fragments are related portions of the molecule.
   
   
5. Molecules like prions can sometimes produce unusual results. There are a lot of mammals that can be studied, especially primates. In some cases these allow for very distinct evolutionary patterns.
   
6. Keep notes on the BW codes used for the species. This will make identifying the organisms on the different branches a little easier.
   
7. Use the view records button to find more information about the organisms. Their classification and other bits of information can be gleaned from this tool.
   

Comparing the amino acid sequences using clustalw.
After collecting the related species information for a particular protein you should be returned to the session page with your choices now showing. Check the boxes of related molecules you want to use and click the CLUSTALW box. A new page appears and allows you to alter some parameters of the Workbench and change the pull down menu for the guide tree display to rooted and unrooted trees . Submit this to the workbench.

Clustalw is an alignment tool that compares the sequences of the amino acids in the chosen organisms and aligns them by their portions of similar sequence order. The next page shows a few things, the aligned amino acids that are similar will be colored in blue (fully conserved) or green (semi-conserved) for the parts that are most similar. Conservation is a property that shows identical or nearly identical portions of the molecule. These tend to show important structural areas of the protein that need to be maintained for proper function. Conversely the areas of dissimilarity are where the evolution happens. Due to their location they don’t tend to affect the function, yet show where changes have occurred. These changes are what help determine the shape of the trees.


Note: If you notice any apparent oddities to the trees, check to see if the same portions of the molecule are being compared (alpha vs. beta, tentative analyses vs. validated). Also, when using several proteins don’t clustalw them together.

Rooted trees are projected from the side rather than the ground but if you take the monitor and lay it on its side they will look just fine. (JUST KIDDING! LEAVE THE MONITOR ALONE!) There is actually a wraparound effect that can occur with two groups on opposite sides of the tree actually being closely related. This is related to the problem of trying to generate trees for a 2-Dimentional monitor screen from what is more of a 3-Dimentional analysis of branching patterns.

Unrooted trees are pictured like a branching tree as if you were looking at it from above. This can sometimes provide the greatest insight into the divergence of groups of organisms.

Generating a composite cladistic or phylogenic tree.
After gathering the various trees generated by Biology Workbench program try to find similar organisms between these data.

Generating a composite tree means you need to place the differing analyses for the organisms into one tree. To combine the trees you produced with BW look for similar organisms in the diagrams. Overlapping the one data set into the other is one way to make a bigger tree. However, there are problems that may arise, some organisms may appear on differing branches in different protein analyses. They may be linked in one but on a completely different branch in another. What might cause this problem and what might be done to resolve the discrepancy to make the most accurate tree?

CLASS ACTIVITIES
Students will work in groups in an attempt to identify the evolutionary history of a particular group of organisms based on the protein sequences of various molecular analyses. By utilizing various features of Biology Workbench (BW) the students will try to show how similar organisms are related using rooted and un-rooted phylogenic trees.

Procedure:
1. Form groups of 2 or 3 students to do the investigation.
2. Students will choose from either a list of organism groups or a group that is approved by the instructor of similar appearing or taxonomically related organisms.
3. Identify each of the organisms by their scientific name.
Note: It is some times easier to see which organisms are available from the lists obtained in the databases and look up the scientific names through the View Record(s) feature in BW.
4. Using at least 3 different protein compounds appropriate for their organisms (ex. Hemoglobin for higher animals, myoglobin in muscle, enolase is good for most organism comparisons, cytochrome c in all organisms, etc.), create and print (or save to disk) the BW evolutionary trees for those organisms and the respective proteins. Note: Some organisms of the group may not have a particular protein analysis and so other proteins will need to be used to gain insight into the evolutionary past. Also, some "trees" may be in conflict and an average picture may need to be created.
5. Based on these trees create a composite tree on poster board with the pictures obtained from the different sources at hand (books, magazines, internet etc.)

Possible animal and plant groups for study (some may be similar in appearance though not necessarily related by analysis):

1. Canines (Dog, Wolves, Coyote, Dingo, Hyena, African Wild dog, Foxes, etc.)
2. Felines (House cat, Lynx, Tiger, African lion, Mt. Lion, Jaguar, Leopard, Panther, Cheetah, etc.)
3. Bears (Polar, Black, Kodiak, Grizzly, Panda, etc.)
4. Trees (Red oak, White oak, Sugar maple Norway maple, Am. elm, Ginkgo, Green spruce, etc.
5. Mollusks (Slugs, Snails, Squid, Octopus, Cuttlefish, Clams, Oysters, etc.)
6. Arthropods (Insects, Crabs, Spiders, Centipedes, etc)
7. Flowers (Roses, Tulip, Tiger lily, Daylily, Carnation, etc.)
8. Any other collections or subdivisions of plants or animals (Specific insects, specific members of a particular taxonomic genera, families or orders, types of ferns, etc)





OPEN ENDED PROBLEMS
Based on the information obtained from the BW the students will make a composite phylogenic tree or cladistic diagram.