 A
Student Perspective
The Use of Bioluminescence in Gene Research
Author: Laurie Keith - Michigan State University
I have been researching fireflies and their bioluminescence
this spring in my Biology class. I learned how the fireflies
give off their light and how they use it in different forms
of communication. To expound upon my studies, I researched
more about the uses of bioluminescence, not only through
fireflies, but also through biology. Because of the bright
green florescent light it gives off, bioluminescence has
been used in many biology labs.
After interviewing Byron Wingerd, post-doctoral research
assistant at Michigan State University, I was able to assemble
the following information regarding thesis work bioluminescence
as a molecular biology tool. Dr. Wingerd is technically a
post Ph.D. researcher in Prof. Whalon’s, my ISB-202
instructor’s, laboratory in the Center for Integrated
Plant Systems. He is funded by the Michigan Life Science
Corrider funds and is working on the induction of plant secondary
defenses by insects and pathogens. His Ph.D. is from microbiology,
but he is in the Cell and Molecular Biology program, working
in a laboratory in Biochemistry. His thesis research involved
looking at the transcriptional regulation of the inducible
prostaglandin endoperoxide synthase-2 (Cox-2) gene (NSAIDS
and Cox-2 inhibitors, 2001) in response to lipopolysaccharide
(LPS) in a murine macrophage cell line.
What Byron is doing is a very complex process to students
like me, but is just a simple ongoing process for him. He
is using bioluminescence as a tool to study gene expression.
Fireflies have a protein called luciferace, and a substrate
called luciferin. The luciferace protein oxidizes the luciferin
substrate, in an Adenosine triphosphate (ATP) dependent reaction,
giving off a green florescent glow. Dr. Wingerd was able
to utilize this process to study the regulation of the Cox-2
gene.
The Cox2 gene is involved in inflammation when someone sprains
their ankle, or has arthritis. When the Cox2 gene is expressed,
the prostaglandins are made, which cause the inflammation
and swelling. Dr. Wingerd is trying to find a way to turn
off the switch and stop the Cox2 gene from being turned on,
which would prevent the swelling and pain associated with
traumatic tissue damage. In the cases of arthritis and the
spraining and twisting of ankles, one does not want this
Cox2 gene to be turned on, because it will swell and hurt.
If Byron can understand all the parts of the promoter that
are necessary in turning that gene on, he can take one of
those binding proteins and inactivate it, so that it can’t
start inflammation, or at least down regulate it.
Using recombinant DNA technology, the promoter of the Cox-2
gene was cloned in to a plasmid that also encodes the luciferase
gene. The promoter acts as an “on and off” switch
for the luciferase gene. He takes a plasmid, which is a small
circular part of DNA that replicates in bacteria, and sticks
it in ecoli (bacteria), to make many copies if this piece
of DNA (Plasmid Processor Homepage, 1995). After making many
copies, he then takes this DNA and injects it into the mouse
macrophage cell.
If the macrophage turns on the gene for Cox2 through the “on
and off” switch, it will also turn on and start producing
a large amount of luciferace protein, which is easy to detect
in cheimluminescent enzymatic assays because it starts glowing.
Using this system the critical regions of the promoter can
be identified by identifying mutations in the promoter, which
prevent the induction of the luciferase gene (Wingard, 2002).
It is somewhat difficult to detect the Cox2 gene versus
detecting the luciferace protein. To detect the luciferace
protein, he gives the cell a signal and waits a certain amount
of time, and then takes the cells just as they are and rips
them open. He then adds a substrate, luciferin and ATP, to
see if it glows in the dark. The more it glows in the dark,
the more the Cox2 gene is turned on. What makes it glow is
an electron transition as the luciferase enzyme converts
luciferin to its oxidized oxyluciferin form. Instead of looking
to see if the protein is there, all he has to do is see if
it glows in the dark or not.
The stimulant he was working with was LPS, a lipopolysaccharide
found on the outside of gram negative bacterial cells. Your
body is able to detect it, and when it does, you have an
inflammation response. Murine macrophage cells respond to
LPS by producing high levels of the Cox-2 protein. This means
a lot to his research because this high level of response
makes them an ideal model for studying the inflammatory response.
According to the “Basic Concepts in Biology Text,” which
genes are being expressed depends on the type of cell, it’s
moment-by-moment adjustments to changing chemical conditions,
which external signals it happens to receive, and it’s
control systems (Starr, 214) The “systems” that
control the expression of genes consist of molecules. For
instance, regulatory proteins influence transcription, translation,
and gene products. Some components (Cox-2) are activated
or inhibited by signaling molecules (LPS) (Starr, 214). When
he gave the macrophage the stimulant LPS, it gave the cell
a signal that activated the gene to get turned on, which
activated the promoter to turn on, making more luciferace
proteins.
To show the different intensity levels of the light, he
did an experiment. He put some of the cell lysate into some
wells of a 96 well plate. He then put the plate into an instrument
called a luminometer. The detector sits over top of the well
and the instrument injects a little bit of the luciferase
reagent. If the enzyme is present then the substrate gets
oxidized, and will light up. The detector is a very sensitive
photomultiplier connected to a computer, which automatically
records the light intensity (Photon Detection, 1998). This
process takes only a half-hour for results. Other processes
take 8 hours or more, but because of the use of bioluminescence
he can get results in 30 minutes! This technique is much
better and more efficient.
Data base searches help to identify potential consensus
binding sites. By preparing serial deletions of the promoter,
where binding sites occur, the minimal region of the promoter
can be identified. The more he cut off to a certain point,
the less it would glow in the dark, and the switch would
not work. What he was looking to find was which one of these
spots or sections were important to the interaction of the
binding of proteins. If he found that important section,
then it would increase in glow, and the luciferace gene would
be induced.
By identifying this section, he was able to understand which
region of the promoter was essential for upregulating the
transcription of Cox-2. He then made mutations in this piece
of DNA, to make the on and off switch not work. Using this
strategy he can tell which mutations change drastically in
their amount of luminescence, using the bioluminescence as
an indicator of whether or not the gene is turned on or off.
In order to figure out which of these putative binding sites
are essential for the promoter, it is important to be able
to take away the binding site for it. He wants to know which
parts are important binding sights to turn off protein synthesis,
so he can turn the Cox2 gene off and stop it from being transcribed.
Why does he want to do this? I’ll tell you why.
By turning the gene off, it would prevent the swelling and
pain associated with the traumatic tissue damage. Stated
before, in the cases of arthritis and spraining and twisting
of ankles, one does not want this Cox-2 gene to be turned
on, because it will swell and hurt. Therefore, if Dr. Wingerd
finds an important protein binding interaction, makes a small
mutation or change to where it cannot bind anymore, then
the gene will be turned off, and there will be no glow.
This means wonders to the medicine world. Right now, we
have painkiller drugs such as Vioxâ, CelebrexTM , Ibuprofin,
and aspirin that inhibit the Cox2 gene (Cox-2 Inhibitors,
2000). After about 4 to 12 hours, the medicine will ware
off and the patient will have to take more medicine to reduce
the pain. If the transcription of Cox-2 can be modified or
stopped pharmacologically, then it would prevent Cox-2 from
being made in the first place. In the case of arthritis,
the patient could control the transcription of Cox-2 so that
after a few days, there will be no inflammation. Since the
Cox2 gene is not turned on, then the prostaglandins that
cause inflammation would not made, providing relief inflammatory
pain. With the development of the new drugs, it could prevent
the enzyme from being made; compared to the drugs we have
now, that only stop the enzyme for a short period of time.
With the use of bioluminescence, this research is possible.
Without it, Dr. Wingerd, and other researchers out there,
would have never been able to figure out a way to possibly
turn off this Cox2 gene. If they do find an accurate way
to do this, and make this new medicine, this will turn over
a new leaf for the medical field. Pain relief will be much
simpler, and swallowing pills will be an image of the past.
Soon drugs such as Ibuprofin and aspirin will only be a secondary
answer to our pain relief.
Brooks, Peter M. Cox-2 Inhibitors. 2000. University of Queensland,
Brisbane. 24 Mar. 2002. <http://www.australianprescriber.com/magazines/vol23no2/cox2.html>
Kivira, T. Plasmid Processor Homepage. 12 Dec. 1996. Department
of Computer Sciences and Applied Mathematics, University
of Kupio, Finland. 2 Apr. 2002. <http://www.hytti.uku.fi/~oikari/plasmid.html>
Selective Cox-2 Inhibitors. 1997-2000. The Drug Monitor.
1 Apr. 2002. <http://www.home.eznet.net/~webtent/coxi.html>
Sherf, Bruce A., Keith V. Wood, Luminometry for In Vivo
and In Vitro Reporting of Firefly Luciferase. Nov. 1993.
Promega Corporation. 2 Apr. 2002.< http://www.promega.com/pnotes/44/sherf/sherf.html>
Shwartz M.D., Allan B. NSAIDS and Cox-2 Inhibitors. 2001.
MCP Hahnemann University. 18 Mar. 2002. <http://webcampus.med.mcphu.edu/cme/medicine/nsaids/history.htm>
Starr, Cecie. Basic Concepts in Biology. USA: Brooks/Cole,
2000. Pg. 241
Wingard, Byron. Personal Interview. 2 Feb.
2002
Wingard, Byron. Doing What? Michigan State University. 12
Feb. 2002. <http://www.msu.edu/user/wingerdb/doing.htm>
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