Rice endosperm showing GUS expression.
Photo by Lijun Tian, CAMBIA.
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Rice endosperm showing GUS expression.
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In transactivation, upstream activation sequences (UAS) are inserted into plants separately from the transcriptional activators which regulate them. This enables the transcriptional activators to create changes in gene regulation by either inducing or suppressing the expression of certain genes even though they are not linked to the promoters of these genes.
Many investigators have worked with transactivation (see the February 2006 issue of Plant Journal for a recent review), but at CAMBIA our focus shifted to developing a system free of the IP constraints of the systems widely used in Arabidopsis and Drosophila, to enable people to get around barriers to producing technology that is actually deliverable (see Connett and Jefferson 2005).
Use of transactivator sequences presents a method for coordinate expression of multiple genes in a pathway. It may also present a means to "work around" patents that block use of many promoters "operably linked" to genes of interest.
Transactivation research at CAMBIA has been done principally in rice (funded by the Rural Industries RDC and in the Rockefeller Foundation funded work of several Ph.D. students, some still being examined by the relevant thesis committees, but available by agreement). Potential applicability extends to many species and we encourage you to contact us if you are interested in using and improving these sequences in your species of interest.
Unfortunately, CAMBIA is no longer able to distribute materials. Please contact us to find out if there are any labs which may be able to share materials with you.
Notes on plant materials:
CAMBIA is
currently completing an inventory for databasing its transactivator lines in
rice, many with molecular analysis showing segregation patterns up to three
generations. Collaborative projects have already been initiated with research
groups in China, Indonesia and Vietnam. We can make seed available from UAS
lines in which the gene expression pattern may be of interest to regulate by
crossing with a transactivator line. If you are interested even before the
inventory is complete or have ideas toward improvements in the database, please
contact CAMBIA.
Transactivation operates from a transcriptional "enhancer" (actually, this could be involved in up-regulation or down-regulation in different contexts) and at least two cassettes containing the following component sequences: in one, a transcription activator (transactivator), and in the other(s), an upstream activation sequence (UAS), a minimal promoter that normally drives no transcription, and at least one gene whose expression is of interest, which can be a reporter gene or trait gene.
Transactivation vectors are inserted into the plant of interest via any method, such as TransBacter™. When the activator is expressed, it releases a DNA-binding molecule. The UAS serves as a binding site for the binding molecule released by the transactivator cassette, and may induce or suppress the expression of the adjacent gene.
The UAS and minimal promoter may be linked to any one gene or more than one gene. If there are multiple genes in the genome that are linked to UAS::minimal promoter cassettes, the act of binding to the UAS induces or represses them in parallel. When a reporter gene is linked to the minimal promoter and UAS, its expression can be used to monitor the expression of the transactivation sequence (1). However, the transcription factor molecule binds to any copy in the genome of the UAS (2), with no requirement that it be linked to the UAS.
Thus the expression by the reporter gene is initially useful to allow a researcher to determine the locations and developmental stages of transcription factor expression within the plant, and when work on a pathway is done, as an internal standard to measure the expression of other such genes with less readily detectable phenotypes.
The transactivation sequence can be introduced into the plant at the same time and on the same construct as a UAS/minimal promoter sequence (as shown above), but most effectively for pathway manipulation the two cassettes can be inserted into the genome separately. One method of doing this is described in our patent application WO 01/21781. For more detail, see the related page Using Transactivation Lines.
Choices about the method will depend on what genes or characteristics the researcher is trying to express. For example, we are currently interested in improving the system for work with bioindicators (sentinel plants).
All constructs are available under a Plant Enabling Technologies BiOS License and the associated BiOS Technology Support Agreement or a BiOS compliant MTA (contact us for more information).
Researchers and those who are attempting to develop traits that involve multiple genes in a pathway may prefer to use the transcription factor and upstream activation sequence (UAS) cassettes (for example pTNTQ60 and pTNTQ61.3) in separate plant lines that can be crossed.
There are a variety of reasons for doing this. One of the most powerful is that multiple genes can be constructed with the same UAS, and whether they are transformed or crossed in simultaneously or at different times, they can be coordinately regulated by a single transcription factor that is transformed or crossed in separately.
This strategy may be useful to explore gene functions or identify potentially useful promoters using a line that contains a new transcription factor insertion ("transactivation line") without altering genetic expression in that line. In such instances the cross could be made to any line with an upstream activation sequence (UAS) coupled to a reporter gene such as GUSPlus.
Sexually crossing a transactivation line with a UAS 'Gene X' line may also be advantageous when researchers are endeavoring to create a large number of diverse offspring.
Crossing in this way, described in CAMBIA's published patent application WO 01/21785, may be necessary to circumvent constraints in certain jurisdictions on certain patented promoters, that prevent the activation of a promoter by a binding molecule transformed into the same plant.
Where such patent constraints are not in force, obtaining coordinate expression of unlinked genes could also be accomplished by co-transformation with the artificial transcription factor encoded by pTNT.Q.61.3 and constructs containing any genes regulated by the UAS cassette of pTNT.Q.60, or by subsequent transformations of a line already containing the artificial transcription factor encoded by pTNT.Q.61.3.
Full sequences and diagrams of these constructs are available on a page visible to all who have executed a BiOS license. (Anyone else wishing information may write to cambia@cambia.org).
Transactivation can induce the expression of a gene or genes that wouldn't normally be expressed in a particular plant organ or developmental stage. This is referred to as "ectopic" expression.
Transactivation can also be used to suppress expression of certain genes in some plants, plant organs or developmental stages. Transactivation might be used to bind and down-regulate a gene that is necessary for the synthesis of toxic byproducts, for example.
One of the most exciting potential applications of transactivation is the cumulative effect of several improvements within the same plant line. Genes in a biosynthetic pathway may be introduced or up-regulated in a coordinate manner while by-product genes may be simultaneously down-regulated.
After a transactivation sequence has been shown to be active in a particular way, several generations of crosses can be done into lines containing a UAS with various specific genes to create progeny with a combination of characteristics. This approach has been validated throught three generations in Ph.D. student work recently completed at CAMBIA.
Through transactivation-mediated gene expression one possibility of interest to CAMBIA is apomixis - the creation of a crop capable of asexual reproduction. Another is the bioindicator project - the creation of crops that can signal farmers regarding the needs for inputs such as fertiliser, to avoid over-input and waste of resources by enabling timely decisions by the farmers themselves.
Through BiOS-compliant agreements, improvements in the constructs to better accomplish any of these goals can be shared for the benefit of all in the protected commons, while the commercialisation of trait genes used to make products is not restricted by any of these agreements (see BiOS Agreements Frequently Asked Questions).
Manipulation of gene expression, as it is done in most labs, uses multiple technologies which have been patented. Although not-for-profit researchers are rarely subject to infringement actions for using patented technologies, any later improvements or commercially viable developments can be jeopardized by these choices early in the research.
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Rice florets showing anther-specific GUSPlus expression. |
This is because permissions must be obtained from patent holders and licensing and royalty arrangements must be in place before development toward commercialisation can be done. The cost of obtaining these permissions after the fact, when added to field testing and scale-up work, can be prohibitive. Accordingly, sometimes even after years of valuable research work, virtually all public good output or commercialisation is blocked, to the detriment of the global community (see see Connett and Jefferson 2005).
Normally the patent claims for promoters specify that the isolated promoter sequence must be operably linked to a specific gene or genetic sequence. Therefore, many promoters can be used without infringement of the patents when not operably linked to those specific sequences.
This transactivation project has created proprietary tools that are being released under the BiOS license and a BiOS-compliant MTA to the BioForge commons (contact licenses@cambia.org). The tools include an artificial transcription factor with a strongly plant-active DNA binding domain and activation domain, and a vector containing the corresponding upstream activation sequence and a minimal promoter, constructed by CAMBIA (see a 2004 published abstract describing Thach Tran's thesis work; enabling description has been presented in a variety of public fora since 2000 and is in his not-yet-accepted Ph.D. thesis, which we are able to make available together with the vectors to any BiOS licensee on request). This transcription factor/activation sequence pair, not linked, may enable, in many jurisdictions, methods to legally circumvent many existing promoter patents.
By using these constructs under license, a license that includes covenants to maintain the technology available to others and the right to commercialise, researchers will be taking steps to reverse the trend by which the researcher is unable to influence deliverability of good work.
Another means of circumvention is the method of sexually crossing a plant that contains a transactivation sequence with a plant that contains a UAS sequence; offspring containing both vectors may not be infringing on a patent that may preclude researchers from introducing both components into the same plant. See our patent application, WO 01/21781. All CAMBIA technology and know-how related to this work is freely available for use by anyone who agrees to the terms of the BiOS license.
The goal of the BioForge is to create ways to work around patents that are stifling innovation, via collaboration, sharing patented knowledge and unpatented know-how, and making available leveraged access to other essential information and materials for a shared platform of capability to use.
This page is only viewable to all who have executed a BiOS license
*This page is only visible to those who have executed BiOS agreements. Under the terms of these agreements, anything posted on this page should be kept confidential to those who have agreed to the BiOS terms of responsible sharing.*
We will be using this page to link in the thesis work of two Ph.D. students whose work has involved transactivators. Under the terms of the BiOS license what will appear on this page is designated as confidential to licensees, and the links will not download for non-licensees.
Thach Tran developed a new series of trans-activation constructs. He created an artificial transcription factor using the activation domain of one transcriptional activator combined with the DNA-binding domain of another to get very high activity in plants.
Sujin Patarapuwadol tested the stability of trans-activation through up to three generations of rice crosses.
There is a confidential forum linked from this page as a workspace for discussions about the patents covering aspects of trans-activation, for anyone who desires to make observations under an expectation that these would only be seen by others who have agreed to the non-assertion terms of the BiOS agreement.
The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome, please send an email to info@bios.net.