INFORMATION SYSTEMS FOR BIOTECHNOLOGY


December 2005
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:



OPEN-SOURCE AGRICULTURE
C. Neal Stewart, Jr.

Computer software is amenable for duplication, modification, and improvement and therefore has greater utility and value ... DNA as well. Sharing software freely has enabled the open-source movement and has led to numerous innovations in operating systems and products. What about open-sourcing DNA–is that the key to agricultural innovation and feeding an ever-growing population?

One person who thinks so is Richard Jefferson, of GUS reporter gene fame, who is the director of CAMBIA (http://www.cambia.org) and its new offshoot, BiOS (Biological Innovation for Open Society, http://www.bios.net/daisy/bios/15). BiOS and other organizations such as PIPRA (Public Intellectual Property Resource for Agriculture; http://www.pipra.org) are promoting open access to biological innovations targeted to agricultural improvement, especially for crops most important to the developing world.

Currently there are relatively few companies, located mainly (about 75%) in the private sector,1 that hold patents on crucial agricultural biotechnologies; however, Jefferson believes those few companies could be using those patents to "dominate then destroy an industry." Alternatively, he is advocating parallel engineering–that is, the creation of redundant inventions to endow freedom to operate.2 A perfect example of this is the TransBacter system–the ‘new Agro'3–in which bacteria other than Agrobacterium tumefaciens were shown to transfer DNA stably into plant genomes. Indeed, CAMBIA is providing free access to Sinorhizobium meliloti, Mesorhizobium loti, and Rhizobium sp. NGR234 as Agrobacterium alternatives. Much work remains to increase the transformation efficiency provided by these bacteria, but the research is seminal. CAMBIA allows researchers to use these bacteria for free in non-profit and for-profit research and product development; but, in return, the recipient must pledge to make any subsequent improvements freely accessible to others.

Typically when a company makes an invention, it files for a patent and does not freely share the invention–whether it be a promoter, gene, transformation tool, or any other enabling technology. Companies do not share because a patent will allow them to recoup any investment they have made on the invention, as well as secure an exclusive use of the technology for 20 years, when the patent runs out. Of course, they might elect to license the technology to other parties, as DuPont does for biolistic-mediated plant transformation, if it appears that the licensee will not be a direct competitor. Herein lies the difference: status quo is protective and open source is disseminative. The former rests on free market economic forces, while the latter is dependent on altruism and the future gambit that participants will benefit from collective access to improvements

The poster child for open-source agriculture is 'Golden Rice', which is engineered to contain elevated provitamin A content.4&5 The researchers' intentions were to make this rice seed freely available to farmers in the developing world to combat dietary vitamin A deficiency and the childhood blindness that often results. To do so, they literally sweet-talked and shamed patent holders into allowing the technologies, which were restricted by myriad intellectual property rights, to be available, and so enabled a path for Golden Rice to be marketed. Someone looking at the development of Golden Rice might ask the obvious question: if patent holders do not make agricultural biotechnology tools freely accessible, how do researchers like those working on golden rice get them in the first place? The answer is that companies often do make these tools freely accessible for research in a roundabout way. All plant biotechnology researchers have disarmed Agrobacterium, the 35S promoter, and the nptII antibiotic resistance gene in their labs and use them for plant transformation research. These tools get passed around informally from group to group, with the patent holders opting not to block this type of non-profit research. However, it is when the technologies are used in commercial products that patent holders make a stink. Open-source would be more fragrant in that regard.

The question then becomes, would open-source really be effective in facilitating commercialization and technology transfer that could help the poorer farmers of the world? There is no simple answer. Perhaps smaller companies would benefit from open-source biotechnology, because they could then have a chance to play. However, substantial federal and state regulations for products of agricultural biotechnology pose significant barriers for all but the largest of companies. Simply put, many companies wishing to sell transgenic seed will not likely have the millions of dollars needed to meet current regulatory requirements. Regardless whether products were intended for cultivation in the U.S. or Pakistan, for example, the initial regulatory costs might easily outweigh the intellectual property costs. We can hope, however, that regulatory hurdles will eventually be relaxed, as biosafety concerns are assuaged by time and track records–at least for agronomic traits that are becoming very familiar stories in agricultural biotechnology. Value added and output traits might not see regulatory slack for decades. Still, for certain traits and crops, open-source and open-access agriculture has merit.

PIPRA is comprised of a consortium of non-profit research groups–mostly located at land grant universities–committed to making "agricultural technologies more easily available for development and distribution of subsistence crops for humanitarian purposes in the developing world and specialty crops in the developed world" (http://www.pipra.org). Thus, the PIPRA model can be distinguished from open-source: PIPRA seeks to collaborate with member institutions on intellectual property policy, organize a public-sector intellectual property database, and develop shared technology packages.6 PIPRA has positioned itself to work with university technology offices to walk the tightrope between optimal commercial return and optimal humanitarian benefit. Whereas open-source is focused on cumulative improvements, PIPRA seeks to package public sector-derived technologies to support innovation in crops grown in developing countries.

In addition to regulatory hurdles and organizational constraints, there is a dearth of public science that can enable agricultural innovation relevant to the poorer farmers of the world. Even if the TransBacter system is not proved to infringe on broad patents, there are other crucial pieces of science and technology needed to commercialize transgenic plants. For example, genes of interest, promoters, and marker genes are all needed to provide a complete package for freedom-to-operate scenarios. And then there are other technologies that might be useful on the regulatory end, such as recombination tools that could deliver transgenic plants enabled to contain their transgenes in space and time.

Solving these problems starts and ends with funding, since there is little else to limit the willing and able plant scientists with records of innovation. Other than a few foundations to support biotechnology for developing countries and the meager public funding in the developing countries themselves, there is little interest in funding the research needed to make a real difference. The plant ABC transporter that confers kanamycin resistance to transgenic plants7 was discovered serendipitously en route to completing a project funded by the U.S. Army. I personally would much rather work for explicit humanitarian purposes at the onset of a project. Indeed, a concerted effort to discover other selectable markers and promoters is needed, along with deep pockets for funding. The same is true for gene containment technologies. Indeed, commensurate with funding, it would be desirable to guarantee that research would be available for open-source or open-access platforms before discoveries are made. A few researchers are already engaged in this paradigm. PIPRA is involved in the production of a plant transformation vector, with components obtained from the public domain and its member institutions, which is intended for royalty-free use for humanitarian purposes.

Whether the CAMBIA/BiOS, PIPRA, or some other organization ultimately succeeds in facilitating increased access to the biotechnological tools of agricultural science, the beginning of the 21st century should be noted for initiating these important steps toward agricultural equity between North and South. Who knows what will eventually work, but matters as weighty as political instability, mass starvation, and world economic depression could weigh in the balance of the eventual outcome.

Acknowledgement. Thanks to Sara Boettiger for reviewing and discussing an earlier version of this article.

References
1. Graff G D et al. (2003) The public-private structure of intellectual property ownership in agricultural biotechnology. Nat Biotechnol 21, 989-995

2. Herrera S (2005) Profile: Richard Jefferson. Nat Biotechnol 23, 643

3. Broothaearts et al. (2005) Gene transfer to plants by diverse species of bacteria. Nature 433, 629-633

4. Ye X et al. (2000) Engineering provitamin A (b-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303-305

5. Potrykus I (2001) Golden rice and beyond. Plant Physiol 125, 1157-1161

6. Atkinson R C et al (2003) Public sector collaboration for agricultural IP management. Science 301, 174-175

7. Mentewab A & Stewart C N Jr. (2005) Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants. Nat Biotechnol 23, 1177-1180

C. Neal Stewart, Jr.
University of Tennessee, Dept. of Plant Sciences
Knoxville, TN
nealstewart@utk.edu



A CONCEPTUAL FRAMEWORK FOR THE DESIGN OF ENVIRONMENTAL POST-MARKET MONITORING OF GENETICALLY MODIFIED PLANTS
Olivier Sanvido

In 2004, 97% of worldwide commercially cultivated, genetically modified plants (GMP) were grown in five countries, i.e., USA, Argentina, Canada, Brazil, and China. None of these countries requires legally binding post-market monitoring (PMM) activities, or they are limited to very specific areas of concern, such as insect resistance monitoring of Bt maize, for example, in the United States. The regulatory frameworks of these countries recognize GM products that have received regulatory approval for commercialization if the products are substantially equivalent to comparable products with a history of safe use, and thus, do not present a greater risk. Environmental PMM or long-term health surveillance are therefore not considered necessary.

In regards to the possible environmental effects of GMPs, the principle of substantial equivalence is not followed in Europe, and a precautionary approach is chosen instead. Everyone who intends to commercially grow GM crops in the European Union (EU) is obligated to present a PMM plan to identify possible adverse effects on human health and the environment, which could arise directly or indirectly from the released GMP.1 To date, no EU-wide consensus on how to design such PMM programs has been defined, although monitoring concepts are currently being developed in several European countries. There is an urgent need for conceptual frameworks and guidance on how PMM programs should be planned and performed. The aim of our study was to develop a conceptual framework containing structures and procedures that could be used to implement such PMM programs.2 This framework should represent a pragmatic approach for feasible PMM programs that allow the assessment of possible environmental effects during commercial cultivation of GMP.

Procedure for the approval to cultivate GMPs in Europe
Each approval for commercial cultivation of a specific GMP has to be preceded by case-by-case risk assessments of potential adverse effects on the environment. Furthermore, the introduction of GMPs into the environment should generally be performed according to a step-by-step principle, which means that the scale of GMP releases can only be increased if a risk assessment of the preceding step has estimated an acceptable risk for the next step.

We established a scheme that clearly presents and distinguishes the different phases and activities of development and commercialization of a GMP (Fig. 1). Pre-market risk assessment (PMRA) is limited to the phase prior to approval for commercial cultivation, whereas PMM is limited to activities related to the commercial cultivation of GMPs. According to EU legislation, PMM is composed of two separate programs with different aims, i.e., case-specific monitoring (CSM) and general surveillance (GS).

Principles of environmental monitoring programs
We felt a strong need for a clear definition of the specific functions and differences of CSM and GS, as well as for a definition of what tasks should be accomplished in each program. In order to clearly distinguish the differences between the two programs, we analyzed the general principles of existing environmental monitoring programs.3,4 Based on these general principles, CSM and GS can be more clearly defined, and their respective limits can be identified:

1. Case-specific monitoring is intended to assess whether GMP-related adverse effects on the environment occur.1 It is based on specific risks that a particular GMP could present. CSM can be regarded as the continuation of the investigations performed during PMRA where defined hypotheses on possible anticipated effects are tested. The hypotheses can be confirmed or rejected after a defined period of time, after which CSM can be terminated (Fig. 1). As CSM is performed in close relation to the cultivation of a certain GMP, it should be possible to draw conclusions about the causes of detected changes. The gain of knowledge may lead to new questions, which have to be answered in specific risk assessment studies. CSM helps to reduce remaining uncertainties, and its results may influence the PMRA of new GMPs with comparable properties.

2. General surveillance is intended to detect unanticipated adverse environmental effects that were not identified and considered during pre-market risk assessment.1 Results obtained from GS cannot be linked to any specific attributes of GMP cultivation, since the program provides a general assessment of the state of the environment, independent of any preconception. It can provide information on exceptional environmental changes, and possibly provide basic information to forecast the likely development of the environment. GS is not designed to determine the cause of possible environmental changes, as a multitude of factors could be involved. If environmental changes are observed, and it is considered likely that the cultivation of a specific GMP has caused them, the causality will have to be determined through specific risk assessment studies (Fig. 1).



Figure 1: Stepwise procedure of ecological risk assessment during the life cycle of a specific genetically modified plant (GMP). Pre-market risk assessment and post-market monitoring are two distinct phases during the evaluation of possible risks of GMPs. The two phases are separated by the approval for commercial cultivation, which represents a significant step in this process (ERA = environmental risk assessment).

Many existing monitoring programs face the problem of providing only limited information on quality and changes of the environment, because their purposes have not been exactly defined.4 We have identified clear conceptual differences between CSM and GS and propose to adopt separate frameworks when developing either of the two programs.2 Common to both programs is the need to put a value on possible ecological effects of GMP cultivation.

Challenges for post-market-monitoring programs
According to EU legislation, consent for commercial cultivation is given for a ten year period, after which the results of PMM and any other new information have to be presented in an environmental risk assessment to the competent authority in order to allow renewal of the consent. The time period chosen for PMM may be shorter than the ten year period given for the consent, but it could be extended beyond the consent period for detection of delayed effects.5 However, it is important to consider that the life-span of modern crop varieties may be shorter than the ten year period. For example, during the 1980s the average life-span of an oilseed rape cultivar was about ten years, but dropped to three years by 1997. It might therefore become difficult to perform CSM over an extended period of time for a specific GMP variety.

In CSM it may be difficult to relate environmental effects unambiguously to a specific GMP or its cultivation. All crops and all farming systems cause environmental impacts, and the effects detected could have been caused by factors other than the GMP. Intensification of agriculture, for example, has a range of impacts on biodiversity with widespread decline throughout many groups of organisms associated with farmland in Europe.6 An unbiased evaluation has to consider a reference system that displays the environmental effects that may occur without the cultivation of GMPs. Case-specific monitoring requires a comparable cropping system without GMP as a parallel control, where both crop systems are evaluated in parallel over the same time period. However, such a paired comparison might become difficult in practice, if, for example, the non-transgenic control is not cultivated in the same region or in a comparable agricultural landscape. An additional difficulty could arise from differences in crop management techniques for GM and non-GM plants. For example, GM herbicide-tolerant crops may be best managed by using a no-till strategy, while this technique may not be advisable for cropping systems based on conventionally bred plants. If a parallel control with a comparable cropping system without GMP is not possible, environmental impacts of GMP cultivation need to be compared based on general information of effects caused by current agricultural practice. While the cultivation of Bt maize, for example, may have weak effects on non-target arthropods, the use of a synthetic insecticide can significantly affect a large number of non-target arthropods.

Conclusions
Environmental post-market monitoring of genetically modified plants represents a new challenge for farmers, the agricultural industry, scientists, and regulators, since comparable environmental monitoring programs have not been established for conventional crops. However, the challenge to obtain information on the state of the environment is not new, and underlying principles have been established. Although these monitoring programs were originally designed for general environmental protection, the inherent principles also remain valid for environmental PMM of GMPs. The existing experience documented in the literature shows that monitoring programs require defined aims and a rigid structure in order to provide the desired information. Competent authorities will have to make decisions on maintaining consents for GMP cultivation based on the results of PMM. Case-specific monitoring and general surveillance have to be designed and implemented according to a pragmatic and realistic approach in order to be feasible. Competent authorities can support this approach by applying comparable valuation criteria for effects of GMP cultivation and for effects caused by current agricultural practice. We believe that our conceptual framework will be of assistance to industry, researchers, and regulators when assessing possible environmental effects of GMPs during commercialization.

Acknowledgements
We thank the Swiss Agency for the Environment, Forests and Landscape for partial funding of this study.

References
1. European Community (2001) Directive 2001/18/EC of the European Parliament and of the council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC, European Parliament and the Council of the European Union

2. Sanvido O, Widmer F, Winzeler M, & Bigler F (2005) A conceptual framework for the design of environmental post-market monitoring of genetically modified plants. Environmental Biosafety Research 4, 13-27

3. Hellawell JM (1991) Development of a rationale for monitoring. In Monitoring for Conservation and Ecology, B. Goldsmith, Editor. Chapman and Hall: London, 1-14

4. Vos P, Meelis E, & Ter Keurs WJ (2000) A framework for the design of ecological monitoring programs as a tool for environmental and nature management. Environmental Monitoring and Assessments 61, 317-344

5. ACRE (2004) Guidance on best practice in the design of post-market monitoring plans in submission to the advisory committee on releases to the environment, Department for Environment, Food and Rural Affairs - Advisory Committee on Releases to the Environment

6. Robinson RA Sutherland WJ (2002) Post-war changes in arable farming and biodiversity in Great Britain. Journal of Applied Ecology 39, 157-176

The original publication is available at http://www.edpsciences.org/ebr.

Olivier Sanvido
Agroscope FAL Reckenholz
Swiss Federal Research Station for Agroecology and Agriculture
CH-8046 Zurich, Switzerland
olivier.sanvido@fal.admin.ch



APPROACHES TO MINIMIZE VARIATION OF TRANSGENE EXPRESSION IN PLANTS
Katleen Butaye

The development of genetic transformation methods has led to numerous applications of transgenic plants, including the improvement of certain crop traits, the high-level production of valuable proteins, and the use of transgenic plants as a tool to elucidate gene function. However, considerable variation of transgene expression is often observed within populations of transgenic plants transformed with the same transgene construct and under identical transformation conditions. This inter-transformant variation of transgene expression highly complicates phenotype analyses and the production of commercial crops with stable and predictable transgene performance. To reduce cost, labor, and interpretational flaws, multiple efforts are being directed toward achieving stable expression of transgenes with an expected level of expression. Various factors are thought to influence this variation of transgene expression in plants, including inconsistency of transgene copy numbers, the transgene insertion site, and RNA silencing in the host plant. We recently reviewed the current methodologies to minimize inter-individual variation of transgene expression in nuclear transformed plants.1 This review expounds on different transformation methods, recombinase-mediated approaches, gene targeting, optimization of transgene cassettes, viral suppressor genes, and RNA-silencing mutants.

We describe how a widely-used strategy to obtain predictable transgene expression levels is the generation of single-copy transformants, as it is generally believed that the copy number of the transgene influences the level of transgene expression. Several reports suggest that the transgene copy number locus may depend on the transformation method used (Agrobacterium vs. direct), the Agrobacterium strain, the plant species or ecotype, and the explant type of the host plant (ref. 1 and citations therein). Also, Agrobacterium strains that are less efficient in delivering T-DNA, or less favorable transformation conditions, may result in lower copy number of the transgenes. However, to date, it seems that no sound correlations between copy number and transformation methods/parameters can be made.

A more promising method to generate single-copy transgenic plants is the recombinase-mediated resolution of multiple insertions, which also allows site-specific integration of the transgene (reviewed in ref. 2). Typically, this approach involves random integration of a target site, such as the lox site, in the plant genome. Subsequently, single-copy lox-containing transformants are selected, and the new DNA construct is integrated into this genomic target site via recombinase-mediated site-specific integration. Theoretically, this approach yields primary transformants harboring a single-copy insert of the transgene at the predefined locus, thus minimizing the inter-individual variation among transgenic plants. However, so far this method has only been partially successful and efficiency is poor. Furthermore, insertion of the lox site is still random, and ideally, a single copy transgene should be targeted into a highly transcribed region of the plant genome without disruption of existing plant genes.

Gene targeting into a predetermined chromosomal site could be achieved by homologous recombination. To date, however, the moss Physcomitrella patens is the only (lower) plant that is attributed a high rate of homologous recombination, and because the integration of transgenic DNA through homologous recombination is so infrequent in higher eukaryotes, targeting transgenes to a specific position is nearly infeasible. Ample effort is devoted to tackling the problem of the low frequency of homologous recombination in higher plants (not as much as to optimize transgene expression as to facilitate functional analysis of endogenes by insertional mutagenesis of the gene of interest3). Attempts concentrate on rigorous selection methods of successful transformants, improving the efficiency involved in the enzyme machinery for homologous recombination (e.g., the use of recombination hot spots isolated from Escherichia coli or Saccharomyces cerevisiae) for transformation of plants, or the induction of double-strand breaks in DNA. Hence, the application of techniques involving homologous recombination for minimizing variation of transgene expression is far from routine, but recent progress in the field is encouraging.

Other strategies to reduce variation of transgene expression focus on the specific elements of the employed transformation vector, rather than on the transformation method as such. For instance, the employment of suitable promoters and terminators definitely strongly impacts transgene expression. Ample effort has gone into the isolation and characterization of strong, constitutive promoters, as numerous applications of plant biotechnology require constitutive, high-level expression of the transgene.

Generally considered to be strong, constitutive promoters in plants are the widely used p35S, the cassava vein mosaic virus promoter (CsVMV), and plant-derived promoters driving housekeeping genes, including ubiquitin promoters. Furthermore, several research groups have reported strikingly high transgene expression using expression cassettes regulated by optimized 5' and 3' regulatory sequences or using other regulatory elements like intergenic regions of rDNA (ref. 1 and citations therein). Hence, an extensive list of regulatory sequences is at hand when high-level production of proteins is desired, but it is not clear which regulatory sequences or adaptations to constructs are suitable for proper transgene evaluation and high-throughput screening of phenotypes, both of which require low variation of transgene expression. We previously showed that p35S-driven transgene expression in Arabidopsis thaliana is typically characterized by a bimodal expression pattern consisting of 20% high-level expressors and 80% low-level expressors, whereas promoters derived from the mannopine synthase genes yield less variable but also lower transgene expression.4 Hence, one should carefully consider which regulatory elements to utilize for construct design, because optimization or implementation of specific regulatory elements may substantially increase transgene expression levels and reduce transgene expression variability.

In addition to the careful selection of 5' and 3' regulatory sequences to direct transgene expression, the implementation of matrix attachment regions (MARs) in transgene constructs might be considered when low variation of transgene expression is desired. MARs are non-transcribed, A/T rich regions in eukaryotic genomes that by definition have the property of binding partially purified nuclear scaffold proteins in vitro. A number of studies have investigated whether the presence of MARs flanking transgene constructs results in less variable transgene expression (reviewed by ref. 5). Unfortunately, the use of different methods and materials complicate proper comparison of the multiple studies that have been performed on the effect of MARs on transgene expression. In most of these studies, MARs significantly increased average transgene expression levels with limited or no decrease of variability. In some cases, copy-number-dependent expression, enhanced transformation efficiencies, promoter dependency, and subsequent generation effects have been observed (ref. 1 and citations therein). The use of the A element of the chicken lysozyme gene to flank both ends of a transgene cassette significantly reduced the variance of p35S-driven transgene expression in tobacco.6 In wild type A. thaliana, no effect of chicken MARs on transgene expression was observed.4 However, upon transformation of A. thaliana gene silencing mutants, a clear impact of these MARs on transgene expression was observed.7 This suggests that the positive influence of MARs on transgene expression in wild type plants may be suppressed by other factors. Hence, it is currently assumed that MARs can positively influence transgene expression levels and variability in specific experimental designs. It remains to be seen whether this MAR-effect will be generally applicable.

A different way of tackling low transgene expression is the implementation of the current knowledge about the RNA-silencing mechanism in transformation technologies. For instance, the use of viral suppressors of gene silencing has proved to be an efficient strategy to inhibit the negative influence of RNA silencing on transgene expression. As such, co-expression of a transgene and a viral suppressor gene might be an attractive option to reduce variation of transgene expression caused by RNA silencing (ref. 1 and citations therein). Another approach to outwit the negative impact of RNA silencing on transgene expression is the use of RNA-silencing mutant backgrounds as the target of transformation. We recently demonstrated that stable, high-level transgene expression may be obtained using the A. thaliana gene silencing mutants, sgs2 and sgs3,8 resolving the typical bimodal expression pattern obtained for p35S-driven transgene expression in wild type plants. Furthermore, p35S-driven transgene expression remained high and steady in next generation transformed mutants, in marked contrast to the variable expression patterns observed in wild type second generation populations. It was further shown that transgenes flanked by chicken MARs caused a significant boost in transgene expression in the transgenic gene silencing mutants, reaching up to 10% of total soluble protein, whereas no such boost was observed in wild type background. MAR-based plant transformation vectors used in a gene silencing mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants. Although this 'MAR-gene-silencing-mediated' approach was applicable for up to eight different transgenes, using MARs from various origins and gene silencing mutants that were affected in different genes, extrapolation of these results to other plant species remains to be investigated. RNA-silencing-impaired backgrounds in these species may be found through mutational screens or created by use of the hairpin technology to downregulate genes playing a role in the RNA-silencing machinery.

It is clear that predictable transgene expression levels requires further optimization of these methodologies or the development of novel strategies. We believe that one should carefully monitor the progress made in the development of these technologies for immediate implementation in the design of suitable vectors or transformation strategies. Some technologies might be preferred above others, depending on the specific goal of plant transformation.

References
1. Butaye K, Cammue B, Delauré S & De Bolle M (2005) Approaches to minimize variation of transgene expression in plants. Mol. Breeding 16,79-91

2. Ow D (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol. Biol. 48, 183-200

3. Kumar S & Fladung M (2001) Controlling transgene integration in plants. Trends Plant Sci. 6, 155-159

4. De Bolle M, Butaye K, Coucke W, Goderis I, Wouters P, van Boxel N, Broekaert W & Cammue B (2003) Analysis of the influence of promoter elements and a matrix attachment region on the inter-individual variation of transgene expression in populations of Arabidopsis thaliana. Plant Sci. 165, 169-179

5. Allen G, Spiker S & Thompson W (2000) Use of matrix attachment regions (MARs) to minimize transgene silencing. Plant Mol. Biol. 43, 361-376

6. Mlynárová L, Jansen R, Conner A, Stiekema W & Nap J (1995) The MAR-mediated reduction in position effect can be uncoupled from copy number-dependent expression in transgenic plants. Plant Cell 7, 599-609

7. Butaye K, Goderis I, Wouters P, Pues J, Delauré S, Broekaert W, Depicker A, Cammue B & De Bolle M (2004) Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J. 39, 440-449

8. Mourrain P, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel J, Jouette D, Lacombe A, Nikic S, Picault N, Remoue K, Sanial M, Vo T & Vaucheret H (2000) Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101, 533-542

Katleen Butaye
Centre of Microbial and Plant Genetics
Katholieke Universiteit Leuven, Belgium

Correspondence: Bruno Cammue
Centre of Microbial and Plant Genetics
Katholieke Universiteit Leuven, Belgium
bruno.cammue@biw.kuleuven.be


DEXTRAN-PRODUCING TRANSGENIC POTATO TUBERS
P. Janaki Krishna

Plants and plant cells have become increasingly popular as vehicles for the production of therapeutic proteins and peptides, and are efficient bioreactors of recombinant biopharmaceuticals such as cytokinins, hormones, monoclonal antibodies, enzymes, and vaccines. Now, a novel polymer, dextran, can be added to that list. A team of researchers from Wageningen University, The Netherlands, and Stanford University and Columbia University, USA, has recently published a report on the production of the dextran in potato tubers and its effect on starch biosynthesis.

Dextran is used as a food ingredient, in cosmetics as a moisture-retaining ingredient, in hematology as a blood plasma volume expander, and in immunology as a vaccine adjuvant. Commercially, a water-soluble, branched dextran polymer is produced via fermentation of sucrose by the bacterium Leuconostoc mesenteroides. The biosynthetic process is mediated by the dextransucrase DSRS enzyme. It was recently established that the DSRS glusosylation reaction can also be used for the synthesis of new compounds such as oligosaccharide and surfactant derivatives, giving access to novel industrial applications.

In the study reviewed here, scientists used a starch-accumulating crop, potato, to investigate dextran production. Two potato genotypes, cv. Kardal and the amylose-free (amf) mutant, were used for transformation. A binary plant expression vector was constructed containing a mature dextransucrase DsrS gene isolated from Leuconostoc mesenteroides. Insertion of the DsrS gene into the potato amyloplast was achieved by first fusing it to a chloroplast ferredoxin (FD) signal peptide and then complexing that with the highly tuber-expressed patatin promoter. The resulting pPFS binary vector was transformed into Agrobacterium tumefaciens using electroporation. Dextrans were identified by enzyme-linked imunosorbent assay (ELISA) in tuber juices of potato transformants. Semi-quantitative and real-time quantitative RT-PCR was used for expression analysis of DsrS and starch synthesizing genes. Morphological and physicochemical properties of the starch granules were studied.

Results were analyzed for 30 independent transgenic potato clones. Five plants of each transgenic clone were grown in the greenhouse. Transformed plants were denoted as KDDxx and amfDxx, in which D represents the 'DsrS' gene and 'xx' the clone number, and the untransformed genotypes were referred to as 'KD-UT' and 'amf-UT'. Transformants KDD15, KDD4, KDD5 and KDD30 contained the largest amount of dextran, ranging from 1.0 mg to 1.7 mg g-1 FW. In the KDD series, dextran was found in 9 out of 30 tubers (29%). Also the dextran concentration was two times higher in the Kardal genotype (about 1.7 mg/g FW) than in amf transformants. No dextran was detected by ELISA inside the starch granule. In general, results from the semi-quantitative, real-time quantitative RT-PCR and ELISA correlated very well with each other. Dextran accumulation did not affect plant morphology and tuber growth. In the highest expressing close, KDD30, tuber number and yield were significantly decreased, though the accumulation of dextran was not correlated with these factors. However, starch granules morphology and DsrS expression are correlated. The percentage of altered starch granules was the highest in (++) transformants: KDD30 with 16.0+1.0%; KDD5, 11.0%+1.0%; and KDD4, 10.3+0.6%. However, in the (-) and (+) class transformants, the frequency of altered starch granules was much lower, remaining under 7%. Therefore an altered granule phenotype coincides with dextran accumulation. The researchers concluded that dextran accumulation does not interfere with the physicochemical properties and starch content of transgenic starches. Also, expression of key genes involved in starch biosynthesis was not affected by dextran accumulation. Furthermore, no clear changes in chain length distribution, despite the known accepter efficiency of DSRS, were found.

Thus, the study demonstrates the production of dextran in potato tubers and its effect on starch biosynthesis, mediated by the expression of the DsrS gene from L. mesenteroides. As no tuber and starch yield penalties were observed for plants producing dextrans, this method could offer an advantage for commercialization of dextran production in plant systems. This study is the first report illustrating that dextran polymer can be produced in potato tubers.

Reference
Kok-Jacon GA, Vincken JP, Suurs LC, Wang D, Liu S, Visser RG. (2005) Production of dextran in transgenic potato plants. Transgenic Research 14, 385-395

P S Janaki Krishna
Institute of Public Enterprise
Osmania University Campus
Hyderabad, India
jankrisp@yahoo.com



ESTS: WHAT ARE THEY GOOD FOR?
Phillip B.C. Jones

Absolutely nothing. At least, the second-highest U.S. court says that ESTs can lack a use under the standards of patent law.

Fourteen years ago, the National Institutes of Health (NIH) sparked the debate about whether claims to expressed sequence tags should be patentable. The NIH filed three patent applications that covered over 6,000 DNA fragments isolated with Craig Venter's new method for discovering genes. James Watson, who directed the NIH genome project at that time, denounced the plan to patent the ESTs as "sheer lunacy." Other protests emerged from academia and the biotech industry. The NIH dropped the patent applications.

Yet the question remained about the patentability of ESTs. In the absence of clear guidance from the courts, organizations filed patent applications that claimed bits of DNA. On September 7, the Court of Appeals for the Federal Circuit published its decision for In re Fisher, a case that provides guidance. Stephen Walsh, the assistant solicitor who argued the case for the U.S. Patent and Trademark Office, told the National Law Journal that there are probably more than 100 pending EST patent applications likely to be thrown out in light of the decision.

USPTO rejects EST claim across the board
The case began when Dane K. Fisher and Raghunath V. Lalgudi filed a patent application claiming compositions related to nucleic acid molecules from maize leaf tissue pooled at the time of anthesis. The application included a listing of 32,236 EST nucleotide sequences. According to the application, the ESTs can be used in a variety of ways, including: (1) serving as molecular markers for mapping the entire maize genome; (2) measuring the level of mRNA in a tissue sample to provide information about gene expression; (3) providing a source for primers that could be used with the polymerase chain reaction to duplicate specific genes; (4) identifying the presence or absence of a polymorphism; (5) isolating promoters via chromosome walking; (6) controlling protein expression; and (7) locating genetic molecules of other plants.

The original claims covered over 4,000 different nucleotide sequences. After the examiner required restriction to a smaller set of sequences, the inventors chose five, each containing 331 to 429 nucleotides. The applicants pursued the following claim:

A substantially purified nucleic acid molecule that encodes a maize protein or fragment thereof comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 5.

In September 2001, the examiner rejected the claim as lacking utility and for a lack of enablement, a requirement that inventors teach how to make and use the claimed invention. Without a utility, the enablement requirement cannot be met, the examiner reasoned. Monsanto, the party of interest, appealed the rejection to the USPTO Board of Patent Appeals and Interferences.

Monsanto focused on two uses for the appeal: (1) identification of polymorphisms; and (2) probes or as a source for primers. The Board did not find support for either use. In the Board's view, the patent application failed to explain why the claimed ESTs would be useful in detecting polymorphisms in maize plants. "Without knowing any further information in regard to the gene represented by an EST, as here," the Board wrote, "detection of the presence or absence of a polymorphism provides the barest information in regard to genetic heritage." The Board did not see the second use as meeting the utility requirement, because the application "does not attribute any property in terms of plant trait or phenotype to any of the nucleotide molecules set forth in SEQ NO:1 through SEQ NO:5." Without such information, "using the claimed molecules to isolate other molecules, which themselves lack substantial utility, does not represent a substantial utility."

The examiner's rejections based on a lack of utility and enablement remained undisturbed. Monsanto appealed.

Federal Circuit finds claim unappealing
On appeal, the USPTO argued that Monsanto's general and speculative uses fail to meet the statutory standard for utility in the absence of information about the function of genes and proteins that correspond to the claimed ESTs. Academic institutions and biotech/pharma companies filed amicus briefs in support of the USPTO's position.

Chief Judge Paul R. Michel, who wrote the decision of the three-judge panel, explained the Federal Circuit's view of the law. A claimed invention must have both a substantial utility and a specific utility to pass muster under the utility requirement. The substantial utility requirement can be met if the asserted use of the claimed invention offers a significant and presently available benefit to the public. To satisfy the specific use requirement, an asserted use must also provide a well-defined and particular benefit to the public.

The court decided that Monsanto's patent claim failed the substantial utility requirement, because the ESTs only function is as research intermediates that may help researchers isolate the corresponding genes and perform further experiments with those genes. The court also found that the asserted uses fail to meet the requirement for a specific use because "any EST transcribed from any gene in the maize genome has the potential to perform any one of the alleged uses." In other words, the court concluded that the patent application discloses only general uses, not specific uses as required for utility.

"Here, granting a patent to Fisher for its five claimed ESTs," Michel wrote, "would amount to a hunting license because the claimed ESTs can be used only to gain further information about the underlying genes and the proteins encoded for by those genes." The court viewed the claimed ESTs not as an end of a research effort, but rather as "tools to be used along the way in the search for a practical utility." Without an identification of the function of the corresponding protein-encoding genes, the "claimed ESTs have not been researched and understood to the point of providing an immediate, well-defined, real world benefit to the public meriting the grant of a patent."

Two of the three judges on the Federal Circuit panel affirmed the utility and enablement rejections. Judge Randall R. Rader dissented.

"These claimed ESTs have such a utility," Rader wrote, "at least as research tools in isolating and studying other molecules." He analogized the claimed ESTs to a microscope. Both take a researcher one step closer to identifying and understanding a previously unknown structure, he suggested. If a microscope has utility, then so do the claimed ESTs.

Although the Fisher case offers long-awaited guidance about the patentability of ESTs, it also raises a question. Will future Federal Circuit panels side with the majority decision or adopt Rader's view the next time that a case involves a patent claim on a research tool?

Selected Sources
In Re: Dane K. Fisher, Docket No. 04-1465 (September 7, 2005). Available at:
http://fedcir.gov.

MacLean PA. Biotech patent cases may tank. The National Law Journal, page P4, September 19, 2005.

Phill Jones
BiotechWriter.com
PhillJones@nasw.org




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