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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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