A special note to our readers:

INFORMATION SYSTEMS FOR BIOTECHNOLOGY PROGRAM TO END

After twenty years of serving the agricultural and environmental biotechnology research community, the National Biological Impact Assessment Program (NBIAP) grant was not included in the Federal appropriations budget this year. Although the National Biological Impact Assessment Program funding is in the proposed Federal budget for 2009, we do not know at this point if that funding will be authorized. Of course, without continued funding, the ISB program will terminate. Please be assured, however, that we are tirelessly working toward reinstatement of the NBIAP grant. The many letters of support received from our readers over the years continue to be very helpful in that regard.

Through two decades, ISB has provided informational resources to researchers, educators, regulators, and the public to assist in the safe application of agricultural biotechnology. Through the monthly News Report, the greenhouse containment guide, conferences and symposia, field test and other databases, and the web site, ISB has endeavored to be an impartial and unbiased resource on the current use of agricultural biotechnology and on the biosafety and risk assessment considerations of transgenic organisms.

Regardless of the outcome, we at ISB would like to take this opportunity to thank everyone who has supported our program over the last twenty years. We hope we have provided a valuable service that has been useful and informative to the agbiotech community.

With our sincere thanks,

Doug King, Ruth Irwin, and Lori Duis Simmons

Since the commercialization of Bt maize in 1996, the global area planted to maize or cotton varieties expressing Cry proteins derived from Bacillus thuringiensis (Bt) has steadily increased and reached a total of more than 42 million hectares in 2007¹. Several crops expressing novel insecticidal proteins derived from Bt or other sources are currently under development and will be commercialized in the near future.

Similar to conventional agricultural pest control technologies, one risk associated with growing insect-resistant, genetically modified (IRGM) crops is their potential to adversely affect non-target organisms, which include a range of arthropod species that fulfill important ecological functions such as biological control. The potential for adverse effects of IRGM crops on non-target arthropods (NTAs) thus has to be evaluated as part of the environmental risk assessment (ERA) process that takes place prior to the decision to cultivate these crops commercially. The relative novelty of GM crops and the complexity of ERA procedures present regulatory authorities with a challenge in developing appropriate risk assessment methodologies. This is a particularly difficult task in the developing world, where regulatory infrastructure is still being established.

Corn grain is an important component of feed for non-ruminant animals and food for humans. It is an excellent source of starch, but is a poor source of protein nutrition. Protein content is low, usually less than 10% of the kernel mass, and moreover, the quality of maize protein is not ideal. Maize protein is deficient in certain amino acids—lysine, tryptophan, and methionine—that are required by non-ruminant animals (including humans). To remedy these deficiencies, protein supplements are provided to create a well-balanced diet, which adds to the cost of feed and food. Genetic improvements that increase the levels of lysine, tryptophan, and methionine have been actively sought by researchers for the past fifty years. Recently, several approaches involving one or more transgenes have been successful^1,2,3.

A recently published approach³ tests the hypothesis that amino acid balance in maize kernels can be improved by production of a foreign protein to serve as a sink for amino acids. The foreign protein used in this case is the porcine milk protein alpha-lactalbumin, which is introduced transgenically. The transgene contains a maize seed storage protein gene promoter and a synthetic coding sequence optimized for expression in maize. The coding sequence is modified by the addition of a maize signal sequence and an endoplasmic reticulum retention sequence to direct accumulation of the transgene product only to targeted regions in the cell. This transgene design was predicted to result in accumulation of alpha-lactalbumin only in the endoplasmic reticulum of the kernel endosperm, mimicking the pattern of accumulation of the most abundant natural seed proteins in the kernel, the zeins. The authors of this study found that transgenic kernels accumulate a protein that cross-reacts with antiserum raised to alpha-lactalbumin and is consistent with the size predicted for the transgene product.

The key experiment in this work is a comparison between transgenic and non-transgenic endosperm tissue produced on the same ear. In two events evaluated, transgenic and nontransgenic endosperm differ in their amino acid balance, and the difference generally reflects the amino acid balance of alpha-lactalbumin. Lysine content is 29 – 47% higher in transgenic endosperm than in the non-transgenic sibling kernels. In most cases, total protein content is not changed. Kernel characteristics such as density, mass, and seed storage protein content are not altered. Thus it appears that this modification results in a specific alteration to the endosperm amino acid balance that does not detectably disrupt kernel development.

The change in amino acid content, together with the accumulation of alpha-lactalbumin in the kernels, suggests that alpha-lactalbumin may be acting as an amino acid sink. Closer inspection reveals, however, that the level of alpha-lactalbumin in the kernels is not sufficient to explain the change in amino acid composition. Thus it seems that the introduction of alpha-lactalbumin to the kernels creates a sink for amino acids but the nature of the sink remains unclear.

The choice of a milk protein is interesting because it could result in nutritional benefits in addition to improved amino acid balance. Several studies have shown that derivatives of alpha-lactalbumin have benefits such as antimicrobial and anti-tumor activities. It is hypothesized that some of the health benefits enjoyed by nursing infants are conferred by proteins in milk such as alpha-lactalbumin. The nutritional benefits of alpha-lactalbumin have led several researchers to conclude that human alpha-lactalbumin would be a beneficial component of infant formulas. A logical extension of this idea is to include milk proteins from specific farm animals as supplements to their diets, especially when young animals are weaned. By illustrating the feasibility of producing porcine alpha-lactalbumin in corn, the Bicar study enabled the researchers to test the hypothesis that corn containing porcine alpha-lactalbumin is beneficial in swine diets.

One drawback to using a milk protein is its potential for allergenicity in humans. Porcine alpha-lactalbumin has not been evaluated for its allergenic potential. Most milk allergies involve a reaction to bovine beta-lactoglobin protein; however some individuals react to other milk proteins, and some individuals with bovine milk allergies also react to goat milk proteins. A thorough evaluation of the allergenicity of porcine alpha-lactalbumin is therefore a prerequisite for application of this technology. Regardless of the outcome of this evaluation, this work illustrates the important point that it is possible to alter amino acid balance in a predictable way by adding a foreign protein to seeds using genetic engineering.

The challenge of increasing grain amino acids can be thought of in terms of increasing the strengths of either the source or the sink of the amino acids of interest. As shown in the Bicar et al. report, adding a foreign protein to grain may increase the strength of the amino acid sink. Some transgenic approaches that successfully alter lysine content involve manipulation of lysine metabolism enzymes, essentially increasing the strength of the source of lysine. As described by Kirihara et al.⁴, an effective method to improve amino acid balance could involve alterations that increase both the source and the sink strengths for the amino acids of interest. Thus, the pathways for amino acid metabolism would be altered to increase the level of amino acids available for incorporation into proteins, and a sink for these amino acids would be created by manipulation of the seed protein content and/or composition. This approach has been successfully put into practice¹. In this case, the sink for amino acids is created by reducing the levels of the native, low lysine proteins of corn, which presumably results in an increase in high lysine native proteins. The work of Bicar et al. suggests that a foreign protein can create an effective sink for amino acids and would therefore be compatible with this approach as well. A carefully selected protein could not only create an amino acid sink, but could also confer nutritional or other types of benefit on the seed.

1. Huang S, Kruger DE, Frizzi A, D'Ordine RL, Florida CA, Adams WR, Brown WE, and Luethy MH. (2005) High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation. Plant Biotechnology Journal 3, 555-569

2. Houmard NM, Mainville JL, Bonin CP, Huang S, Luethy MH, and Malvar TM. (2007) High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. Plant Biotechnology Journal 5, 605-614

3. Bicar E, Woodman-Clikeman W, Sangtong V, Peterson JM, Yang S, Lee N, and Scott MP. (2008) Transgenic maize endosperm containing a milk protein has improved amino acid balance. Transgenic Research 17, 59-71

4. Kirihara JA, Hibberd KA, and Anthony J. (2005) Method for altering the nutritional content of plant seed. USA Patent 6,960,709

TRANSGENIC WHEAT HAS INCREASED POLYAMINES
Teresa Capell

Importance of arginine decarboxylase in plants
Crop productivity in modern agriculture relies heavily on abundant supplies of water for irrigation. Although drought and salt tolerance genes are present in wheat, breeding for stress tolerance is time- and labour-intensive and complicated by the multigenic nature of stress tolerance and the complexity of wheat genetics. The polyamine biosynthetic pathway in higher plants is a useful model in which to examine the components that affect the levels of intermediates and end products in the pathway. By introducing appropriate transgenes into plants and measuring the effects of transgene products on end product accumulation, we may begin to understand how individual components of the pathway contribute towards their concerted regulation¹.

The polyamines spermidine and spermine, and their precursor putrescine, are ubiquitous in all living organisms and are involved in many diverse physiological, developmental, and biochemical processes. Pyridoxal phosphate (PLP)-dependent ornithine decarboxylase (ODC) is the initial enzyme in the pathway committed to polyamine synthesis. In plants and some bacteria, putrescine can also be synthesized from arginine via arginine decarboxylase (ADC) through the intermediate agmatine. Putrescine is further converted into spermidine and spermine by spermidine synthase (SPDS) and spermine synthase (SPMS), respectively. These enzymes add aminopropyl groups generated from S-adenosylmethionine (SAM) by SAM decarboxylase (SAMDC). In plants, the two alternative pathways appear to have specific roles in growth and development. While ODC appears to be implicated in the regulation of the cell cycle in actively dividing cells and meristematic zones, ADC is the primary enzyme for putrescine synthesis in non-dividing elongating cells, secondary metabolic processes, and in cells under various stresses².

Arginine decarboxylase is a low copy number nuclear gene that lacks introns in the sequences described to date. Several ADC cDNA clones have been isolated and characterized from various species. Whereas in some plants a single gene encodes ADC, in the Brassicaceae family at least two paralogues exist in all members studied to date except for the basal genus Aethionema. In Arabidopsis, protein sequences derived from ADC1 (U58851) and ADC2 (AF009647) genes show 80% homology; however the activities of these enzymes differ. Much of the difference between ADC1 and ADC2 protein amino acid sequences is at the N-terminus, suggesting that the subcellular location of the two proteins might be different. Polyclonal antibodies raised against tobacco ADC (AF321137; 99% homology to the tobacco ADC2 AF127241) detect ADC protein in all plant organs analyzed: flowers, seeds, stems, leaves, and roots; however, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplasts. These results suggest that the intracellular location of ADC in plants might account for different roles for the enzyme in different locations. Thus the notion was advanced that chloroplastic ADC might be involved in photosynthesis, whereas the nuclear form may play a role in cellular signalling³.

Engineering the polyamine biosynthetic pathway in wheat
Wheat (Triticum aestivum L.) is a staple crop for about 35% of the human population. Its great adaptability to varied climatic conditions makes it one of the most widely cultivated crops, with a short growing season and a good yield per unit area. Breeders have produced disease-resistant, drought-tolerant (to a certain degree) and high-yielding varieties using conventional methodology. However, wheat productivity has declined as a result of deteriorating soil conditions, the quality and quantity of available water for irrigation, and general environmental degradation. Consequently, increases in productivity have fallen below the rate of population growth.

One of the major targets for wheat improvement is drought tolerance. Our group is interested specifically in elucidating the role of polyamines in abiotic stress tolerance in cereal crops. We generated transgenic wheat plants expressing an oat (Avena sativa L.) ADC cDNA driven by the maize 1 ubiquitin (Ubi) promoter and first intron⁴. These plants accumulate up to 2-fold putrescine, spermidine, and spermine in leaves. The two-fold increase in the three polyamines measured in leaves of primary transformants is maintained in the T1 generation, thus confirming the heritable nature of polyamine accumulation in transgenic wheat plants expressing the transgene.

Whereas an increase in polyamine content is rare in rice leaves, such increases are more common in seeds. For example spermidine and spermine levels are significantly increased in seeds of transgenic rice plants expressing Ubi:DsSAMDC⁵. Multiple independent transgenic wheat lines expressing Ubi:AsADC accumulate up to 7-fold putrescine, 1.5-fold spermidine, and two-fold spermine in seeds, compared to wild type plants. These levels are maintained and even enhanced in progeny.

Results from our earlier studies in rice and also our current investigation in wheat demonstrate that less metabolically active tissue, such as seeds, accumulate higher levels of polyamines. Our results are in line with experiments in which metabolites such as pre-vitamin A and pharmaceutical antibodies accumulate at high levels in seeds of rice, wheat, and pea⁶. It is not surprising that higher levels of accumulation occur in seeds, because these storage organs are dormant or certainly less metabolically active compared to vegetative tissue. The above examples show that this behavior is not limited to small molecular weight metabolites. Rather it is more general, extending to the accumulation of recombinant proteins, which in extreme cases can form paracrystalline structures in the endosperm⁶.

The wheat genome contains at least two arginine decarboxylase paralogs
Genomic characterization of the transgenic wheat plants indicated high homology between the oat and wheat ADC. Digestion with several enzymes and hybridization with a cloned partial wheat ADC probe confirmed the presence of at least two ADC genes in the wheat genome. Only two ADC genes have been cloned from monocotyledonous plants to date—the first from oat (X56802) and more recently a second gene from rice (BAA84799). Chattopadhyah et al.⁷, upon digestion of rice and oat genomic DNA with a specific enzyme and subsequent probing with a 498-OsADC probe, observed two distinct molecular species in both rice and oat genomic DNA blots. An ancestral ADC gene appears to have been duplicated early in the origin of the Brassicae family, thus yielding two paralogs. These two different ADC1 and ADC2 genes have been described in Arabidopsis. Although the two genes share 80% homology in their amino acid sequence, they exhibit different expression patterns: ADC1 is expressed constitutively, whereas ADC2 is mainly expressed in cauline leaves and siliques and is induced by different abiotic stresses. Two ADC paralogous genes have been characterized also in Pringlea antiscorbutica, Nicotiana tabacum, and Malus sylvestris L (crabapple).

Using the gene-specific probe for TaADC, we detect two transcripts of ca. 2.6 and 3.1 kb in wheat. The transcripts are expressed in leaves and roots, with the 2.6 kb species exhibiting higher steady-state accumulation. No significant changes are measured in levels of steady-state TaADC (long or short mRNA species) or in TaSAMDC levels in transgenic plants expressing the AsADC gene. It is therefore apparent that the heterologous transgene operates independently of its wheat homologs, and consequently any increase in polyamine content is due to expression of the transgene alone. Some evidence exists for post-transcriptional and post-translational regulation of ADC in plants. TaADC might generate two different forms of the transcript through alternative splicing. Alternative processing of a transcript is common in animal cells, where expression from a single gene may produce protein variants that differ in function, tissue specificity, or sub-cellular localization. There are also reports of alternative splicing in plants. For example Zhang et al.⁸ described the cloning of three cDNAs from apple (MdSPDS-1, -2a and -2b) encoding SPDS. MdSPDS-2a and MdSPDS-2b originate from SPDS2 by alternative splicing and are differentially regulated in a tissue- and developmentally-specific manner.

In conclusion, we generated and characterized a transgenic wheat population expressing AsADC. In the course of this characterization, we detected the presence of multiple ADC homologs in wheat, coinciding with gene duplication in other plant species with complex genomes. This transgenic wheat population will be useful in studies to elucidate further the role of polyamines in drought tolerance and also to ascertain differences and similarities between rice and wheat in their general response to abiotic stresses. Such studies are important because they will allow us to determine whether polyamines are general response mediators that enhance tolerance of plants to abiotic stress.

1. Capell T, Christou P (2004) Current Opinion in Biotech. 15, 148-154

2. Capell T, Bassie L, Christou P (2004) Proc. Natl. Acad. Sci. of USA 101, 9909-9914

3. Hanfrey C, Sommer S, Mayer MJ, Burtin D, Michael AJ (2001) Plant J. 27, 551-560

4. Bassie L, Zhu C, Romagosa I, Christou P, Capell T (2008) Mol. Breeding DOI 10.1007/s11032-007-9154-2

5. Thu-Hang P, Bassie L, Safwat G, Trung-Nghia P, Capell T (2002) Plant Physiol. 129, 1744-1754

6. Stoger E, Williams S, Keen D, Christou P (1999) Trans. Res. 8, 73-82

7. Chattopadhyay MK, Gupta S, Sengupta DN, Ghosh B (1997) Plant Mol. Biol. 34, 477-483

8. Zhang Z, Honda C, Kita M, Hu C, Nakayama M, Moriguchi T (2003) Mol. Genet. Genomics 268, 799-807

If a tree falls in the forest and nobody hears it, does it make a sound? Sure. If a document is placed on an Internet-accessible site, is that document prior art? Well, maybe.

SRI International, Inc. sued Internet Security Systems, Inc. and Symantec Corporation for infringement of four patents that relate to the monitoring and surveillance of computer networks for intrusion detection. The district court granted the defendants' motion for summary judgment that the patent claims lacked novelty in light of a document called "Live Traffic Analysis of TCP/IP Gateways." SRI appealed the decision, contending that the Live Traffic paper did not qualify as prior art.

The Live Paper controversy began when the Internet Society posted a call for papers on its website. All submissions were to be made via electronic mail with a backup submission sent by postal mail. One of SRI's inventors, Phillip Porras, attached a copy of the Live Traffic paper to an email sent to Dr. Bishop, the Program Chair. As a backup, Porras said that SRI would make a copy of the Live Traffic paper available on a SRI website for about one week, and he included the FTP address for the document. Over a year later, SRI filed its patent application on the invention described in the Live Traffic paper.

Under US law, a claimed invention must not have been described in a printed publication more than one year prior to the date of filing the patent application. Courts have decided that a publication has been "printed" if it is accessible to a person who exercises reasonable diligence to locate it. The district court judge decided that the Live Traffic paper had been publicly accessible via the FTP site, which destroyed the novelty of SRI's patent claims.

The Federal Circuit disagreed. A 1978 case, Application of Bayer, concerned alleged prior art in the form of a graduate thesis stored in a university library. Although three faculty members knew about the thesis, the document had not been catalogued or placed on a publically available shelf. The thesis was not a "printed" publication, because a normal search would not have rendered the work reasonably accessible even to a person who knew about its existence.

The Federal Circuit viewed the uncatalogued thesis stored in a library to be similar to the Live Traffic paper stored on the FTP server. The server lacked an index or catalogue for meaningful research, and the directory structure did not identify the location of papers or explain the mnemonic structure for files. Moreover, the record indicates that, outside of SRI, only Dr. Bishop knew about the availability of the Live Traffic paper.

"The Live Traffic paper, like posters at a vacant and unpublicized conference," wrote the Federal Circuit, "was available by being posted,' but available only to a person who may have wandered into the conference by happenstance or knew about the conference via unpublicized means." The Federal Circuit sent the case back to the district court to reexamine the public accessibility of the Live Traffic paper.

In its decision, the Federal Circuit cited a 2004 case, In re Klopfenstein, which concerned posters displayed during two professional conferences. The posters were printed publications, because their entire purpose was public communication. Now, take a step back. Suppose that somebody took notes about a poster and did not make the notes available to the general public. The notes would not be prior art. Yet they could acquire a life of their own to destroy a patent, or even four patents, as Bayer discovered.

Time Bomb in a Notepad

Over 20 years ago, Plant Genetic Systems, a predecessor of Bayer Bioscience, used the Agrobacterium tumefaciens technique to transform plant cells with a fragment of a Bacillus thuringiensis toxin gene. The genetically engineered plants synthesized a truncated version of a Bt toxin. The company submitted a patent application on Bt toxin-producing, genetically engineered plants, which matured into a family of patents including US Patent Nos. 5,545,565, 5,767,372, 6,107,546 and 5,254,799. The patents claim chimeric genes comprising a truncated Bt toxin gene; plant cells and plants that synthesize insecticidal protein; and methods of transforming plants with chimeric Bt toxin genes.

In December 2000, Monsanto filed a declaratory judgment action in the Eastern District of Missouri seeking an assertion that MON 810 YieldGard® products did not infringe the '565, '372, '546, and '799 patents, and that these patents were invalid and unenforceable. The defendant counterclaimed, alleging infringement of claims in each patent.

The district court granted summary judgment to Monsanto, and held that all four patents were unenforceable due to inequitable conduct, that certain patent claims were invalid, and that the '565 patent was not infringed. On appeal, the Federal Circuit reversed the decision and returned the case to the district court.

Back in Missouri, the defendant (now, Bayer) dismissed all claims that MON810 infringed the '799, '372, and '546 patents. The case proceeded to trial on the '565 patent. Judge E. Richard Webber held a four-day hearing on the inequitable conduct allegation. In August 2006, the judge released a 99-page opinion in which he concluded that inequitable conduct rendered the '565 patent unenforceable. Judge Webber also found inequitable conduct in the prosecution of the '799, '372, and '546 patents, and held those patents unenforceable. Bayer appealed.

The Federal Circuit examined the basis for the inequitable conduct allegation. During the prosecution of the '565 patent, the patent applicant had disclosed as prior art an abstract by Dr. Wayne Barnes entitled "A Bifunctional Gene for Insecticide and Kanamycin Resistance," which had been prepared for a poster to be presented at the First International Congress of Plant Molecular Biology in Savanna, Georgia, in 1985. The abstract described a chimeric gene comprising the "first half" of a Bt toxin gene combined with a kanamycin resistance gene. The chimeric gene could be inserted into Agrobacterium T-DNA. Barnes speculated that, "This plant gene should express the insecticide and kanamycin resistance from the same promoter."

In 1994, a patent examiner rejected all patent claims as obvious over various prior art references including the Barnes abstract. According to the examiner, the abstract provided motivation to genetically engineer plant cells with a truncated Bt toxin gene. The patent applicant argued, among other things, that the abstract failed to show that the chimeric gene would work in plants. "Moreover, the Barnes et al. reference is not enabled since it is stated therein that the fused gene may' be inserted into T-DNA and that the plant gene 'should' express the insecticide and kanamycin resistance from the same promoter," the applicant argued. "But no concrete evidence is provided."

However, one of the patent applicant's employees had attended the 1985 conference and had taken notes about Barnes' poster. In addition to details on the construction of a Bt toxin chimeric gene and expression vector, the notes revealed that the Bt toxin fusion protein functioned as an insecticide, and indicated that Barnes had transformed a plant with a chimeric gene construct. The notes had been circulated among officials and employees of the company, including at least one member of the intellectual property department. The company had not submitted the notes to the patent examiner.

To declare a patent unenforceable for inequitable conduct, a district court must find by clear and convincing evidence (1) that a patent applicant breached the duty of candor and good faith to the US Patent and Trademark Office by failing to disclose material information, or by submitting false material information, and (2) that the patent applicant acted with an intent to deceive the patent office. Judge Webber had decided that the notes, if they had been disclosed to the patent examiner, would have contradicted that patent applicant's arguments about the Barnes abstract. Concluding that the patent applicant had made "a deliberate decision to withhold the known highly material reference with the specific intent to deceive or mislead the PTO examiner," the judge declared Bayer's four patents unenforceable due to inequitable conduct.

The Federal Circuit agreed that failure to submit the notes renders the '565 patent—the only patent asserted by Bayer—unenforceable. What about the other three patents that Bayer no longer asserted against Monsanto? Bayer argued that the district court lacked jurisdiction to make a decision about these patents. But Monsanto had filed a request for attorney fees at a time when Bayer accused the company of infringing all four patents. That request was still pending a decision, the Federal Circuit noted. The district court, therefore, had jurisdiction to determine whether inequitable conduct rendered the three unasserted patents unenforceable.

Monsanto Company v. Bayer Bioscience, N.V., 2006 U.S. Dist. LEXIS 97254 (August, 28. 2006).