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Terminator Technology

By Alan Bruzel

The whole point of using Terminator technology is to prevent a farmer from propagating a plant containing an engineered gene – a gene for pest resistance, for example. The transgenic plant manufacturer doesn't want the grower to continually propagate an expensively engineered, pest-resistant plant. If you are the manufacturer, you want the grower to buy seed from you every time he plants. Terminator technology ensures that plants raised by a farmer will be infertile. Here is one way to accomplish this feat; several methods are outlined in United States Patent 5,723,765, "Control of Plant Gene Expression," issued to Melvin Oliver et al. on March 3, 1998.

A good way to ensure infertile seed production is to see to it that a toxic gene is activated during the final stages of seed development. You may use the promoter from a gene produced late in seed development, such as the aptly named Late Embryogenesis Abundant (LEA) gene. (A promoter is a DNA region adjacent to a gene that allows activation of that gene.) Attach this LEA promoter to a gene that produces a toxic product, for example, the Ribosome Inhibitor Protein (RIP) gene. (Also aptly named, as inhibition of ribosomal activity leads to cell death.) You now have a toxic gene that is expressed only in the latter phase of seed development.

During late embryogenesis, when seeds are in their final stage of production, the RIP gene would be activated, yielding sterile seeds. Unfortunately, a plant harboring the above genetic construct would produce sterile seeds for both the manufacturer and the farmer. Before every planting, more genetically engineered plants would have to be made anew in the laboratory – a rather expensive undertaking. It is much better to devise a way of having the plant internally produce the above construct from some innocuous precursor.

Having a long segment of "blocker" DNA between the LEA promoter and the RIP gene allows plants to enter embryogenesis without the risk of producing sterile seeds. The interposed blocker DNA segment prevents synthesis of a functional RIP gene transcript.

Now, you have a construct that never kills its seed. This doesn't solve the problem for the manufacturer, who wants to propagate his plants, while preventing the farmer from doing the same. The manufacturer needs to make the plant remove the blocker DNA and, at the same time, attach the LEA promoter to the RIP gene. In other words, the manufacturer wants to be able to control when his transgenic plants perform the following reaction, which we shall call Reaction One.

The manufacturer, of course, wants this reaction to occur in the farmer's plants, not in his stock.

There is an enzyme called recombinase that will carry out the above reaction. It requires two small pieces of a specific DNA, known as recombinase recognition sequences, to be placed on each end of the blocker DNA. The new construct looks like this:

Now, in the presence of recombinase, Reaction One will be carried out. But, there are two problems:

1.) The transgenic plant needs to be retrofitted with an appropriate recombinase gene. The CRE/LOX system of bacterial viruses produces a recombinase (known as the CRE protein) that recognizes specific stretches of DNA (known as the LOX sequences), and splices out any DNA that happens to lie between those specific recognition sites. Using this system allows you to perform Reaction One. Unfortunately, this brings us to problem two.

2.) You cannot willy-nilly carry out Reaction One. If you simply put a recombinase gene in a transgenic plant that carries your promoter + blocker + lethal gene construct, whenever you grow the plant, it will produce sterile seeds – a situation you have been trying to avoid.

Remember, you want the customer to grow plants that produce sterile seed. Your plants need to bear fertile seed so that you can grow more of your transgenic product. Therefore, if you put a recombinase gene in your plant, you must hold the recombinase in check until you decide to activate it.

Fortunately, you keep up with the literature, and are aware of the Tn10 tetracycline repressor system. The Tn10 tet system consists of specific DNA sequences (known as tetO) and a gene that makes a repressor protein (known as TetR). TetR protein binds to tetO regions. Placing tetO regions upstream of a gene will inactivate that gene – and keep it inactive – if TetR protein is present. This system is used in biotech applications where one wants to selectively switch a gene on and off. Here's how you make use of the Tn10 tet system.

You attach those tetO DNA sequences to your recombinase gene:

You manufacture another genetic construct containing the gene that makes the TetR protein, and allow this gene to be continuously expressed in the plant cell:

After all of this has been accomplished (now you see why genetic engineering development costs can run high, and why patents are necessary to protect one's investment), you now possess three genes:

1.) A construct with a promoter that is activated only in late embryogenesis, separated from a toxic gene by a stretch of inert blocker DNA that is itself flanked with recombinase recognition sites.

2.) A recombinase gene with upstream tetO DNA sequences. The recombinase cannot be activated if TetR repressor protein is present.

3.) A gene that continually makes TetR repressor protein.

Using techniques appropriate for transferring foreign genes into plants, you put these three engineered genes in your plant. (For simplicity, let's say you inject them into the nucleus of a plant cell, and then let the plant grow to maturity from that one cell.)

If everything was done right, all parts – including the seeds – of the newly made transgenic plant will have these genes.

You may now grow the seeds from this transgenic plant and produce many transgenic plants (acres of them, if you so desire). All will bear fertile seeds because the TetR repressor protein is always being produced, keeping the recombinase gene turned off. Without recombinase, the blocker DNA cannot be spliced out, and the LEA promoter and the lethal RIP gene cannot be fused together to yield seed embryo-killing toxin.

Harvest these seeds and store them well; this cache of seeds will allow you to produce more transgenic plants that will again give rise to more fertile seeds. The rest of the harvested seed you are going to sell.

But – and this is the key step – before selling your transgenic seed to the farmer, soak them in a tetracycline solution. The tetracycline displaces the TetR repressor protein from the tetO DNA sequences upstream of the recombinase gene. With no repressor protein obstructing access to the recombinase gene, recombinase can be synthesized. This enzyme then goes on its merry way, splicing out the blocker DNA, and fusing the LEA promoter to the RIP gene.

When the plants grown from this tetracycline-treated seed reach maturity, the LEA promoter will become active in the maturing seeds, and direct the synthesis of the RIP protein. The RIP protein will stop further protein synthesis in the maturing seed. The result is a mature plant with sterile seed.

You have thus constructed a transgenic plant that will bear fertile seed for you, but will, after a simple treatment, prevent others from propagating a second generation of plants from the seed you have sold them. Terminator technology can thus be used to prevent unauthorized propagation of any plant. Following the lead of the software industry, you too have copy protected your intellectual property.

What the Web Has to Say about:
Terminator Technology

Devilish Seed
Terminator technology as seen through memos from the US Department of Agriculture. From Rob Edwards, in New Scientist. Also, see his article, "End of the Germ Line."

How Terminator Gene Stops Seed Formation
Article by N. Gopal Raj for The Hindu Online reviews the details of terminator gene construction.

How the Terminator Terminates
Primer on the molecular biology of terminator gene technology and scenarios envisioned. From Martha L. Crouch, Indiana University at Bloomington.

National Medal of Technology
Presented by President Bill Clinton to Monsanto scientists for their work in transgenic crop biotechnology. A Special Briefing from Scientific American.

Patent Awarded for Plant Gene Expression
News report on the Terminator gene from Information Systems for Biotechnology.

Rural Advancement Foundation International
Terminator gene technology is antithetical to the position taken by this advocate group for rural societies.

Suicide Seeds
Article by Jeffrey Kluger for Time magazine describing terminator technology and public reaction.

Technology Protection System
Appraisal of terminator gene technology from AGCare, an Ontario, Canada crop producers organization.

Terminator Technology
Reprint of article by Ricarda A. Steinbrecher and Pat Roy Mooney originally appearing in The Ecologist, August 1998.

Terminator Technology for Transgenic Crops
How Terminator works. From Charles Hagedorn, Virginia Cooperative Extension.

Transgenic Plants
Background information of transgenic plant production and its accomplishments. From John W. Kimball.

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