The Biology WorkBench is a web-based tool for biologists. The
WorkBench allows biologists to search many popular protein and nucleic
acid sequence databases. Database searching is integrated with access to
a wide variety of analysis and modeling tools, all within a point and click
interface that eliminates file format compatibility problems.
Suggested Web Browser:
the Biology Workbench was originally developed for Netscape Communicator
or Navigator, up through version 4.7x. Microsoft Internet Explorer (especially
older versions) can be unpredictable when loading the Biology Workbench,
but the latest versions of Explorer seem to work fine. Because we are unable
to force Internet Explorer to open secondary windows with our software,
showing database records and reading help pages can be a bit clumsy. Nonetheless,
most Biology Workbench operations *should* work within Internet Explorer.
Before
beginning, open a second web browser window so that you may
view this tutorial at the same time you are working with the Biology
Workbench.
It is recommended
that you not print out these pages unless absolutely necessary. This
tutorial has been designed as an interactive lesson, and
this advantage is negated if it is not done on the web. |
TUTORIAL (20
- 40 minutes)
In many of
the tutorials the beta chain of human hemoglobin has been used as an example.
In the following tutorials a very similar protein will be used. Myoglobin
is an oxygen binding protein which is very similar to hemoglobin.
However it occurs as a monomer in the muscles, rather than as a
tetramer
in erythrocytes.
The muscle
tissue of whales carries particularly large amounts of this protein; consequently
the muscles are able to absorb large amounts of oxygen. This is one
reason that whales can stay beneath the surface of the ocean without breathing
for nearly an hour. Many people erroneously think that whales are
simply holding their breath. In fact, whales may dive to 1000 feet where
the pressure on their bodies is so extreme that their lungs actually collapse!
Thus, all the oxygen they need is stored in the myoglobin of their muscles.
A key component
of a-globin,
b-globin
and myoglobin is iron, which has a high affinity for oxygen. Because
oxidized iron is red (think of rust) myoglobin and muscle is red as well!
The red color of steak in the supermarket is due to myoglobin. If
you ever see a documentary of the Inuit (Eskimos) eating raw whale, notice
that the meat is so dark with myoglobin that it is brown.
Since all vertebrates have muscle,
they should all have myoglobin as well. This tutorial compares
the differences in amino acid sequence in the myoglobins of humans and
ten other animals.
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Click
on image to see full-size! |
Register
for Biology Workbench.
Open the Home
Page for Biology Workbench V.3.2.
Select
"set up a free account", as shown by the blue arrow in the
thumbnail. Fill in the blanks and then click on the button that reads "Register".
After you have
successfully registered, follow the link to the Biology Workbench. Scroll
down the page, until you find a series of buttons, as shown in the
thumbnail to the left.
Click on
the "Protein Tools" button. |
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Import
the amino acid sequence for human myoglobin.
In order to do any kind of BLAST search, a query sequence must be obtained
in FASTA format. This is then copied and pasted into Biology Workbench. |
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BLASTP
search for homologs to human myoglobin.
Blastp searches
in designated protein databases such as PIR or SWISSPROT for matches to
a query sequence (either DNA or protein).
The user is
permitted to select one or more databases to compare. Since human myoglobin
was selected, BLASTP will find all of the animals, plants, even bacteria
and viruses, that have similar sequences. The protein sequences are
ranked in descending order of bit score, so you can see what is the closest
(evolutionarily) to humans. Myoglobin is an oxygen transport protein, and
is generally found in muscles - consequently you probably won't see any
matches with plants, bacteria or viruses. But, if you search other proteins,
like Enolase or Aquaporins, you may find matches with humans. |
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CLUSTAL
W
Clustal W
is a multiple alignment algorithm. It calculates the best match for
the selected sequences, and lines them up so that the identities, similarities
and differences can be seen. The results are displayed as sequences
of letters.
-
If proteins
are being aligned, each letter represents an abbreviation
for a different amino acid.
-
If DNA
is being aligned each letter represents an abbreviation
for a different nucleotide.
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Boxshade
Boxshade presents
the results of the alignment to show sequences which are identical, similar
or different.
The same proteins
from closely related species will be very similar to each other (such as
human and chimpanzee myoglobins). They will have the same amino acids
at most positions - these are said to be conserved.
The same proteins from two species which are not closely related will not
be similar to each other (such as human and whale myoglobins).
They will have different amino acids at many positions - the sequence is
not
conserved at these positions.
In some cases
where the same protein is being compared from many species (such as in
our present example) the amino acid present at a certain position in the
sequence may be the same for most species, but different
in a few. In this case, the amino acid sequence at this position
is neither "conserved" or "not conserved". Instead we speak of a
"consensus
sequence", which is the amino acid present
in most cases. |
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DRAWTREE
and DRAWGRAM
The demonstration
that proteins are frequently similar in many species begs for an explanation.
These two algorithms use the sequence differences calculated by CLUSTAL
W to generate graphics which illustrate phylogenetic trees and genetic
distance ( see below for an explanation of these terms), and provide insight
into the evolutionary divergence of the species.
The biological
interpretation of the similarities and differences in the amino acid sequences
between species is that these changes represent evolutionary differences.
Two proteins which are very similar to each other (such as human and chimpanzee
myoglobins) will have the same amino acids at most positions - these are
said to be conserved.
When the amino acid sequences are "highly conserved" the two species are
considered to be evolutionarily close to each other. The extent
of conservation is therefore a measure of genetic
distance and allows us to retrace the course
of evolution which can then be depicted as a phylogenetic
tree. |
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EXERCISE
(20-40 minutes)
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Open the
Save the web page to your desktop.
-
Launch
Microsoft Word. Open the web page from inside the Word application.
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Save as
a Microsoft Word file. Type your answers into the document,
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E-mail
your answers as a Microsoft Word document, or hand in
a hard copy, as directed by the Instructor.
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