INFORMATION SYSTEMS FOR BIOTECHNOLOGY


July 2001

COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:
Transgenic Food Allergies: The CDC Report On Cry9C
Emerging Technologies: The Minimum Use of Force in Plant Genetic Engineering
An Iron Rice that Survives the Alkaline Test
Web-Weaving Plants
Modifying the Sheep Genome by Gene Targeting
Seeds of a Canadian Judicial Conflict: An Update
Upcoming Meetings



TRANSGENIC FOOD ALLERGIES: THE CDC REPORT ON CRY9C

The global marketing of GM food has been dealt a blow following reports of allergic reactions to StarLink corn, which was detected in corn food products last fall. (See "Outcry Over Cry9C," ISB News Report, March 2001.) Further public distrust of transgenic crops is likely to be fueled by these allegations, in spite of a recent Centers for Disease Control (CDC) report countering the claims that StarLink was responsible for the allergic reactions.1

This "StarLink event" is helping to create a climate in which biotechnology companies increasingly feel compelled to convince the public that food from transgenic plants is essentially identical to that from traditional crops, and poses no greater risks. Public concern over food allergies in general is also making it difficult to allay the fears about the safety of GM foods. Public awareness of food allergies becomes evident during a trip to the grocery store. A label on a box of instant cake mix warns of an allergy risk because the ingredients contain wheat and freeze-dried egg. A can of beans provides a similar precaution because it was prepared with peanut oil.

Investigations of any and all potential food allergy risks associated with GM food are vital for consumer protection. The US EPA and FDA regulatory agencies are responding to public fears about GM foods by providing increasingly close scrutiny of GM-derived commodities. Few traditionally grown foods and consumer products receive this intensity of inquisition.

Cry9C is a protein in StarLink corn that is being scrutinized as the potential allergen. This insecticidal protein, produced naturally by Bacillus thuringiensis subspecies tolworthi, is a variant of a number of Bt toxins, including the commercially used Cry1A. Bt toxins work by binding to specific receptors on insect midgut cells, causing lysis and ultimate decay of the insect's digestive tract. Bt proteins are host specific and do not bind to vertebrate cells. According to the CDC, the Cry9C protein shares several molecular properties with proteins that are known food allergens, which is given as a reason the EPA did not license StarLink corn for human consumption.

A large number of proteins, as well as other organic compounds, including complex carbohydrates, terpene-derived compounds, and simple aromatic molecules, are known to induce allergic reactions. Eggs, milk, peanuts, soybeans, and wheat lead the list of foods causing allergies in infants and young children. Adults are more likely to show allergies to crustaceans, eggs, fish, mollusks, peanuts, tree nuts, and wheat. Researchers working with the augmentation of these and related compounds in GMO foods must take into consideration safety concerns and market resistance to any resulting products intended for human consumption.

The incidence of food allergies in the human population is low, approximately 1% for adults and 5% for infants. It is estimated that about 7.5% of the population has reported some type of food allergy or sensitivity. These data are not conclusive, however, and percentages may be exaggerated by conditions mimicking food allergies such as food insensitivities, mild food poisoning, chemical hypersensitivities, reactions to food additives, and allergic reactions to molds or pollens.2

In their investigation, the CDC established that 28 of the people who filed adverse event reports (AERs) with the CDC after eating corn products containing the Cry9C protein had experienced a true allergic reaction, unrelated to any other medical condition. The human allergic response produces IgE antibodies to the offending antigen, which can be detected in blood serum; consequently, the CDC initially developed an ELISA test for Cry9C-specific IgE antibodies. Coded serum samples were analyzed from three groups of people: the 28 individuals who reported experiencing an allergic reaction to StarLink; people reported to be highly sensitive to a large variety of allergens; and historically banked serum samples collected before Cry9C entered the food supply. Their study could not confirm a link between Cry9C and the production of detectable amounts of Cry9C-specific IgE. However, the CDC report provided a carefully worded conclusion, stating, "Although our results do not provide any evidence that the allergic reactions experienced by the people who filed AERs were associated with hypersensitivity to Cry9C protein, we cannot completely rule out this possibility, in part because food allergies may occur without detectable serum IgE to the allergens."

In summary, the CDC did not exhaustively resolve the issue of allergenicity to Cry9C. The CDC's guarded conclusions still leave the EPA with the responsibility to decide how to regulate GMOs containing the Cry9C protein and related compounds, and the wary public to decide, once again, whether to feel reassured or apprehensive about eating GM food.

Sources

1. Centers for Disease Control. 2001. Investigation of human health effects associated with potential exposure to genetically modified corn. A report to the US Food and Drug Administration from the Centers for Disease Control and Prevention. http://www.cdc.gov/nceh/ehhe/Cry9CReport/executivesummary.htm

2. US Food and Drug Administration. 1994. FDA Consumer: Food Allergies—Rare But Risky. (Updated, 1997.) http://www.cfsan.fda.gov/~dms/wh-alrg1.html

Brian R. Shmaefsky
Department of Biology and Environmental Sciences
Kingwood College
brian.shmaefsky@nhmccd.edu



EMERGING TECHNOLOGIES: THE MINIMUM USE OF FORCE IN PLANT GENETIC ENGINEERING

The global agricultural biotech industry is based upon the constant innovations that emerge from many laboratories scattered around the world. In the early days of the biotech industry, a climate of constant experimentation and novelty led to products that have now reached farms, stores, and hospitals. Outside the context of the lab, these products have prompted a public debate unanticipated by most biotech workers. Public confidence is now the most important factor in the continuing growth of commercially-driven genetic engineering. As a nascent industry matures, so must the technology on which it draws. The next phase of technology development for this industry is to methodically improve and render routine the fundamental technologies, bringing a new precision and greater predictability to the powerful approaches available today.1

Comprehending the complexity of natural systems
The finest examples of powerful yet precise control of biological processes are found in living organisms, whose systems, after millions of years of evolution, are well-honed, robust, adaptable, and capable of rapid response, yet are also fail-safe, highly redundant, self-monitoring and -repairing, and subject to both automatic and executive control or veto at multiple levels. Biotechnology exploits this amazing resource by performing relatively tiny yet significant amendments. One example is the use of genes, none of which have been derived de novo from human ingenuity but are all based on a multi-million year research program carried out by the biosphere. As basic scientific knowledge grows, the manipulations possible will become more exact, the downstream effects more predictable, the products improved, and the customers reassured. When dealing with complex, intricately interacting networks, such as genomes and metabolomes, it is preferable to cooperate and coax rather than to co-opt and commandeer. By taking this approach, the next generation of agbiotech products will be able to provide more to producers and consumers, yet possess a greater `substantial equivalence' to nature's successful experiments.

Plant biotechnology often requires the use of various imprecise methods of transformation to introduce additional genetic material. These processes cause severe changes to cell metabolism by disrupting existing architectures or by activating defense mechanisms designed to cope with entirely different assaults. Methods that release cells from the restraints of higher orders of hormonal control (i.e., cell culture, a prerequisite for some transformation systems) can cause wholesale and detrimental changes in metabolism via somaclonal variation, as most probably occurred in the examples most frequently cited by the anti-GM movement.

Plants can also prevent the expression of virally introduced genetic material via methylation of DNA, although this can perturb the normal regulation of other genes. Such changes in the chemistry of DNA in turn activate transposons, which propagate throughout the genome with disruptive effects on all systems. This phenomenon can be exploited as a tool for functional genomics but is generally undesirable if a novel plant is to be considered substantially equivalent to an existing food crop.

Undesirable outcomes also arise from the method of DNA introduction (which mimics pathogen attack) or from the random insertion of the transgene into sensitive areas of the genome, often many times per genome. In particular, the effects of imprecise insertion may not manifest themselves in early generations since different DNA error-checking mechanisms are activated during growth, reproduction, embryogenesis, and development. These outcomes impact on the time and dollar costs of any transgenic program. One strand of current research aims to reduce these effects by working within and alongside existing processes in the cell.

Finesse not force
A recent paper2 demonstrates the integration of new biological knowledge with existing plant metabolic systems in a way that reduces the disruptive effect of the genetic modification process. Koprek and coworkers devised a method of hitching a ride on a natural transposon to prevent the methylation of introduced DNA. Their aim was to overcome the `silencing' observed in later generations caused by methylation of the transgene, which can occur in more than 50% of the transgenic plants in any one experiment.

To achieve this, Koprek and coworkers devised a method that locates a single copy of the transgene in the genome away from crucial genomic and metabolomic structures. The group created two lines of modified barley using conventional transformation technology: one line carried a particular transposase (the enzyme responsible for the movement of the transposon) not native to the plant, and the second line carried the transgene of interest flanked by DNA patterns that would cause the transposase to recognize and act upon the transgene. Traditional breeding between these two barley lines eventually resulted in a generation containing a large proportion of plants with single copies of the transgene relocated by the transposase to places on the chromosomes where DNA insertion was more tolerant of the genome. The rate of silencing in subsequent generations was dramatically reduced, though not eliminated. This was probably due to other, unaddressed issues such as the chemical composition of the inserted genetic information and its new location.

By piggybacking on the evolutionarily-optimized transposon-mobility system, Koprek and coworkers were able to reduce the disruption experienced by the plant cell when undergoing transgenesis, and thereby achieved a considerable improvement in the efficiency of the transformation. In addition, the relocation of the inserted DNA ensured separation of the transgene and the transposase in many of the progeny, permitting easy elimination of this publicly unacceptable, second genetic element by non-transgenic breeding methods. In complementary work, Hejnar and co-workers3 recently incorporated DNA patterns that protect the cell's own genes from being switched off by methylation (in this case, highly-methylated DNA signatures) into transgene-delivery systems and thereby prevented suppression of transgenes. As with the work of Koprek and coworkers, integration with existing systems of gene control yields an improved result, with obvious benefits to any program of plant genetic engineering.

Although the mechanism of methylation silencing activation is unelucidated, it is known to be intimately linked with an RNA surveillance mechanism termed Post Transcriptional Gene Silencing (PTGS).4 It is also possible to work with the PTGS system in order to achieve results beyond those achieved by `brute force' approaches to gene expression manipulation. PTGS is present in most organisms, indicating a provenance in deep time, and is apparently preserved in order to prevent unwelcome production of incorrect messenger RNAs, which could disrupt the passage of correct information from genes to proteins5 and act as a brake on viral replication.

PTGS recognizes when a given stretch of genic DNA has been transcribed in the wrong direction (`read backwards'), indicating some error in the regulation of that gene, and ensures the destruction of subsequent transcripts from that gene, whether correct or not. This system is responsible for the useful genetic modification technique called antisensing, the introduction of a new copy of an endogenous gene that is always read backwards. Antisensing results in reduced expression of the native gene but is an imprecise method of altering gene output. For some applications it is desirable to switch genes off in such a way, for example, to increase the storage time of soft fruits such as tomatoes.

Smith and coworkers at CSIRO in Australia have devised a significant improvement to the antisense method that eliminates expression of the native gene completely.6 They achieved this by integrating their antisensing into every important step of the multi-stage protein-production machinery to an unprecedented degree. Commonly, plant genes contain introns, regions of DNA that are excised after transcription by `splicing' machinery before they are translated into proteins. The eukaryotic protein production line begins with the DNA-reading enzymes and ends with the delivery of proteins to their correct destinations within the cell, and the splicing machinery is an integral part of this system. Smith and coworkers showed that a particular antisense construction (two copies of the endogenous gene orientated towards a common center and separated by an intron) could cause the ongoing and complete destruction of all transcripts from the native gene, a level of suppression previously unattained. This 100% level of antisensing is most probably due to the full processing of the construct by the complete assembly line right up to some checkpoint guarded by the PTGS system. Constructs without the intron failed to suppress the endogenous gene completely, presumably because these constructs were not constantly associated with the processing machinery.

In support of this hypothesis, other work by Bourdon and coworkers has shown that, contrary to the textbook orthodoxy, the presence and position of introns can affect the outcome of transgenesis considerably.7 In both cases, an improvement in effect and precision was brought about by increasing the involvement of the transgene and its products in the highly complex and interlinked information-handling and error-checking processes that have evolved in the cell. The efficacy of the genetic modification process was related to the extent that the plant's own processes were undisturbed.

Acceptable genetic manipulation
It is clear from the above discussion that the introduction of novel DNA into a genome involves the concomitant introduction of gene-derived material into other systems, processes, and mechanisms (for example, the introduction of novel protein into the proteome). All such introductions may alter the behavior of the system and, via the multi-level integration of these systems and processes, the whole cell. The latest improvements to this technology now being developed involve greater cooperation with the powerful mechanisms already set in place by evolution. By virtue of this, they afford an unprecedented level of control and precision, coupled with a sensible and desirable reduction in the disruption to the organism that is required by the industry and, most importantly, the public. This research theme will evolve further as researchers learn to work alongside other regulated intracellular operations such as chaperoning, controlled protein degradation, and cytoskeletal chromosome migration. It will not be long before, for example, the use of transgene elements such as the 35S promoter from Cauliflower Mosaic Virus to force the subjugation of cellular processes to our whim will be seen as an unnecessary and inelegant use of power, akin to the proverbial use of a sledgehammer to crack a nut.

Sources

1. Elborough KM and Hanley SZ. 2001. Emerging Technologies in Plant Biotechnology. Information Systems For Biotechnology News Report, February 2001, pp 2-4.

2. Koprek T, Range S, McElroy D, Louwerse JD, Williams-Carrier RE, and Lemaux PG. 2001. Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley. Plant Physiology 125: 1354-1362.

3. Hejnar J, Hájková P, Plachý J, Elleder D, Stepanets V, and Svoboda J. 2001. CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression. Proceedings of the National Academy of Sciences (USA) 98: 565-569.

4. Di Serio F, Schöb H, Iglesias A, Tarina C, Bouldoires E, and Meins F. 2001. Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs. Proceedings of the National Academy of Sciences (USA) 98: 6506-6510.

5. Carthew RW. 2001. Gene Silencing by double-stranded RNA. Current Opinion in Cell Biology 13: 244-248.

6. Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG and Waterhouse PM, 2000. Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407: 319-320.

7. Bourdon V, Harvey A, and Lonsdale DM. 2001. Introns and their positions affect the translational activity of mRNA in plant cells. EMBO Reports 2001 2(5): 394-398.

Kieran Elborough & Zac Hanley
Consultants in Plant Biotechnology
New Zealand
Biotech@GreenGeNZ.com



AN IRON RICE THAT SURVIVES THE ALKALINE TEST

Increasing crop yield is critical if we are to feed the world's ever-increasing population without destroying our remaining wild lands. In the developing world, where rice is a major dietary staple, crop yields are often limited by a lack of the micronutrient, iron. A report in the May 2001 issue of Nature Biotechnology indicates that, at least for one crop species, this problem can be solved through molecular biology. Through application of some careful metabolic engineering, Takahashi et al. have now enabled rice to "scavenge" for iron with greater efficiency, allowing the crop to thrive in iron-poor soils.1

Iron deficiency in plants is largely caused by a lack of available iron. At pH values above five, iron in the form of Fe3+ ions reacts with hydroxyl ions in the soil solution, forming insoluble metal oxides that the plant is unable to absorb. As the pH of the soil rises, the amount of iron bound up in this type of complex increases. Therefore, although a soil may have a high iron content, little or none of this iron may be in a form available to the plant. It also means that attempting to cure iron deficiency by adding iron fertilizers is not effective—any added iron will simply become insoluble as well. Since many agricultural soils in arid and semi-arid regions are alkaline in nature, the lack of available iron can have a significantly negative impact on rice crop yields.

Plants can be classified into two groups based on the mechanism they use to survive iron deficiency. The vast majority of plants use a mechanism designated as "strategy I," which involves a combination of proton release (to lower soil pH), reduction of Fe3+ to the more soluble Fe2+ form of iron, and an active Fe2+ uptake transporter. Graminaceous species (grasses), including rice, are "strategy II" plants. Under iron stress, these plants release specific Fe3+-binding compounds known as siderophores, which bind Fe3+ and transport it to the root surface where it is taken up by specific transporters. For most graminaceous species, this is a very effective mechanism, providing the plants with sufficient iron in alkaline soils.

However, this is not the case with rice. Under conditions of low iron availability, rice has been shown to release a far lower quantity of siderophores than other grass species. It is known that there is a close correlation between the amount of siderophores released by a strategy II plant and the ability of the plant to tolerate low-iron conditions, so this suggested to Takahashi et al. a natural target for improvement. If rice plants could be engineered through molecular biology to produce more siderophores, they reasoned, the plants should be better able to withstand iron stress under alkaline soil conditions.

The group had previously cloned two genes, naat-A and naat-B, from barley, both encoding forms of the enzyme nicotianamine aminotransferase (NAAT), a key enzyme in the biosynthetic pathway of siderophore, 2'-deoxymugineic acid (DMA). Takahashi et al. used Agrobacterium-mediated transformation to introduce a genomic fragment from barley, carrying both NAAT genes and their endogenous promoters, into rice plants. The 36 independent transgenic lines recovered were self pollinated, and a number of T2 progeny carrying single copies of the transgenes (as identified through Southern analysis) were selected for characterization.

Interestingly, the researchers found that the two genes, linked to their native promoters, were expressed in the same iron-stress inducible manner as they are in barley. Transcripts for the naat-A gene in roots were only observed when the rice plants were exposed to conditions of low iron availability. The naat-B gene was expressed at a low constitutive level in iron-sufficient roots, with expression levels increasing dramatically during iron stress. Both expression patterns are consistent with the expression of the two genes in root tissue of their native barley. However, some expression was seen for both genes in the shoot tissue of transgenic rice plants experiencing iron stress. Neither gene is normally expressed in barley shoots, even during iron deprivation, but other studies have indicated that rice normally up-regulates the enzymes involved in the DMA synthetic pathway in shoot tissue under conditions of iron deprivation, which may explain the abnormal expression pattern of the introduced genes.

When the roots of transgenic plants were assayed for NAAT activity, Takahashi et al. found a 60-fold increase in enzyme activity under iron stress, as compared to the non-transgenic control plants. However, when they measured the amount of the siderophore end product, DMA, they found that iron-deprived transgenic plants excreted only about 1.8 times the amount of DMA as wild type rice plants experiencing iron deprivation. Nevertheless, even this small increase allowed transgenic rice grown in alkaline soils (pH 8.5) to withstand iron deprivation remarkably better, resulting in suppressed chlorosis, greater overall dry weight, and a dramatic four-fold increase in grain yield as compared to wild type control plants.

The discrepancy between the activity levels of the biosynthetic enzyme, NAAT, and the siderophore end product, DMA, illustrates a common problem with metabolic engineering experiments. Attempting to improve the production of a particular product by up-regulating one or more enzymes in the biosynthetic pathway often fails or produces unexpected results.2 This is because the overall regulation of the entire metabolic pathway is often poorly understood. For example, intermediate compounds in the pathway can be involved in feedback regulation, meaning that increasing the amount of the intermediate by up-regulating the activity of its synthetic enzyme can sometimes shut the whole pathway down. Also, the precursor for the desired compound may simply become exhausted, limiting synthesis.

Another problem is that metabolic pathways are often linked to a number of other pathways, so changing the flux through one pathway can have global implications for the plant. This is particularly a concern with the pathway targeted by Takahashi et al. A significant precursor in the DMA biosynthetic pathway is S-adenosyl methionine.3 This compound is also the immediate precursor of ethylene, a major plant hormone, as well as a number of other compounds. If the other enzymes in the DMA pathway are also up-regulated through genetic engineering, large amounts of S-adenosyl methionine could be used up in the synthesis of DMA, resulting in serious problems with plant growth.

Ultimately, it is difficult to fully assess the impact of any metabolic engineering experiment, but the potential rewards of this approach are so great that it is definitely worth the risk. Iron deficiency is one of the three greatest nutritional problems facing people world wide. Although Takahashi et al. did not assess the iron content in their engineered plants, it seems likely that the nutritional content of these plants may have been improved along with their ability to tolerate alkaline soils. It may be that, when combined with an alternative approach for increasing iron content (such as the introduction of iron-binding proteins promoted by Ingo Protrykus of "Golden Rice" fame4), significant progress may be made toward solving this serious problem.

Sources

1. Takahashi M, Nakanishi H, Kawasaki S, Nishizawa NK, and Mori S. 2001. Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes. Nature Biotechnology 19: 466-469.

2. DellaPenna D. 2001. Plant metabolic engineering. Plant Physiology 125: 160-163.

3. Guerinot ML. 2001. Improving rice yields—ironing out the details. Nature Biotechnology 19: 417-418.

4. Lucca P, Hurrell R, and Potrykus I. 2001. Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theoretical and Applied Genetics 102: 392-397.

Claire Granger
Biologist
alesia_sun@yahoo.com


WEB-WEAVING PLANTS

Over the past few years, many researchers have discovered the value of using plants to produce animal products with therapeutic or marketable value. A variety of animal proteins, such as antibodies and enzymes, are successfully expressed in currently marketed commercial plants grown in culture and in the field.1 Many of these products are either extracted from plants or made available in situ through consumption of the plant or plant product.

Researchers interested in producing therapeutic animal products are beginning to favor the use of transgenic plants as "biofactories." For example, Doug Russell's team at Monsanto Company and Manfred Theisen's lab at Meristem Therapeutics in France have developed feasible and cost-effective models for producing large amounts of therapeutic proteins in cultured plant cells. Other industrial and academic researchers are reporting similar results for extractable compounds in field grown crops.

The large-scale production of spider silk is of interest to many investigators because of its potential value for the specialty textile manufacturing industry. Stephen Fahnestock and coworkers of E. I. Dupont de Nemours & Company (Wilmington, Delaware) have reported inducing yeast to produce spider silk protein;2, 4 however, yeast and bacteria tend to truncate the polymerization process and do not consistently produce the elongated silk strands similar to those secreted by spider spinneret glands. Plants, on the other hand, are capable of processing complete spider silk proteins, also known as spidroins, into long chains.

Following initial success with E. coli, Albert Abbott and Michael Ellison at Clemson University hypothesized that the Golden Orb-Weaver spider (Nephila clavipes) spidroins 1 and 2 could be expressed in plant seeds. Similarily, Udo Conrad's lab at IPK-Gatersleben in Germany has also focused on producing spider silk in plants and is working to express complete spider silk fibers in plants for use in high-strength and novel textiles.3 Conrad claims spider dragline silk has "remarkable mechanical properties" because of its great tensile strength, making it as tough as Kevlar fibers and stronger than steel cables of equal cross-sectional area.

A notable account of silk's high tensile strength goes back to 1881. Arizona physician George Emery Goodfellow noticed that a silk handkerchief was not damaged by a bullet, which was removed during an autopsy after a shoot-out. The bullet tore through the person's clothing and skin before fracturing a bone. The handkerchief wrapped around the bullet without tearing, even after penetrating bone. Goodfellow also documented that silk bandannas were capable of preventing bullets from damaging flesh, whereas cotton and wool materials had no similar effect.

Specialized medical applications of spider silk are employed by Nexia Biotechnologies Incorporation in Toronto, Canada, which currently produces a transgenic spider silk called BioSteel in vitro in goat mammary gland cells. Their intent is to use spider silk as a biocompatible matrix for wound closure and in vascular wound repair devices, hemostatic dressings, and medical device products. The use of spider silk as ligature is not new. Ancient Greek writings record its application to cover and seal bleeding wounds. Use of plants as silk "factories" would prove advantageous for the medical use of spider silk because of the reduced likelihood of spreading concealed mammalian diseases to humans, as is theoretically possible with animal models. Nexia has formed an alliance with Conrad's group at the Institute for Plant Genetics and Crop Plant Research to develop plant systems of silk production.

Conrad and coworkers recently reported using a synthetic spider silk protein gene similar to the one used by Fahnestock in E. coli and yeast2, 4 to investigate spider silk production in plants. Artificial gene segments were synthesized using phosphoramidite chemistry from a cDNA sequence for the spidroin 1 gene found in the Nephila clavipes spider. Conrad created a set of different sized gene constructs to evaluate the optimum structure for proper downstream processing of the silk polymer in vivo and in vitro. Each construct was inserted in an E. coli pUC19 system by organizing various fragments to match the natural spider silk genomic sequence. They also investigated novel silk production by adding sequences from the silkworm moth (Bombyx mori).

The constructs were linked to a CaMV 35S promoter for ubiquitous expression. Both C and N terminal signal peptides provided for retention of the transgenic protein in the endoplasmic reticulum. Sequestering spider silk in ER is favored because of the ease of quantitating and isolating the protein. They used a C terminus c-myc-tag to facilitate protein detection with Western blotting. The expression vectors were then placed into transformation vectors and inserted into tomato and potato leaf disks cultured in kanamycin media. Leaf disks were transferred to soil for this experiment. The spider silk was extracted from ground plant cells using relevant protein protocols involving a series of chilled centrifugation and precipitation steps.

Conrad achieved silk production in both leaf and tuber cells in 31% to 69% of the transformed plants. Some of the constructs achieved a high accumulation rate of silk proteins, exceeding 0.5% of the total soluble protein. Conrad's reported rate of silk production in the plant cells was equivalent to the E. coli system designed by Fahnestock.4 Conrad commented that large-scale silk production in his plant system could exceed the yield currently achieved by E. coli in bioreactors.

A future direction for this line of research includes developing differential expression systems for silk in specific plant structures. Spider silk expression targeted to seeds could be favorable for harvesting and extracting the silk from field-grown plants. Mechanisms for inducing the secretion of spider silk and similar polymers in liquid-cultured plant cells are needed for simplifying large scale manufacturing operations. However, Conrad asserts that "another barrier to the application of such materials is the present lack of appropriate techniques to convert the raw material into manufacturable intermediate products."

These initial successes in plants provide another avenue for the biosynthesis of commercially and medically important polymers. The uses of spider silk produced in plants may surpass the simple medical products and textile fabrications currently being investigated. Artificial genes used for programming self-assembling polymers similar to spider silk can be placed in plants, and the polymers harvested for engineering applications such as liquid-crystalline structures and matrices for microelectronics devices. Bioprocessed polymers can also be used for high-strength and high-temperature adhesives for surface coatings. The push for materials with environmentally friendly product life cycles would benefit from the development of biologically synthesized polymers that can be used and disposed of with little toxicological impact on nature.

Sources

1. Conrad U and Fiedler U. 1998. Compartment-specific accumulation of recombinant immunoglobins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Molecular Biology 38(1/2): 101-109.

2. Fahnestock SR and Bedzyk LA. 1997. Production of synthetic spider dragline silk protein in Pichia pastoris. Applied Microbiology Biotechnology 47(1): 33-39.

3. Scheller J, Gührs K-H, Grosse F, and Conrad U. 2001. Production of spider silk proteins in tobacco and potato. Nature Biotechnology 19(6): 573-577.

4. Fahnestock SR and Irwin SL. 1997. Synthetic spider dragline silk proteins and their production in Esherichia coli. Applied Microbiology Biotechnology 47(1): 23-32.

Brian R. Shmaefsky
Department of Biology and Environmental Sciences
Kingwood College
brian.shmaefsky@nhmccd.edu



MODIFYING THE SHEEP GENOME BY GENE TARGETING

The technique of nuclear transfer has afforded scientists the ability to develop animals, other than mice, with precise genetic modifications. The mutation or deletion of a specific gene by homologous recombination is known as "gene targeting" and is a powerful tool for genetic analysis. In the June 2001 issue of Nature Biotechnology, researchers from the Roslin Institute (UK) have reported their efforts to develop sheep lacking the genes for either alpha(1,3)galactosyl transferase or the prion protein. Although all of the modified lambs died shortly after birth, this report still demonstrated the feasibility of gene targeting in sheep.

The two target genes used in this study, alpha(1,3)galactosyl transferase (GGTA1) and the prion protein (PrP), have important human biomedical applications. The GGTA1 gene plays an important role in tissue/organ transplantation. All animals except Old World monkeys, apes, and humans express the sugar molecule galactose-alpha(1,3)galactose on their cell surfaces. Thus when tissues from these animals are transplanted into humans, the human immune system recognizes them as foreign and mounts an immune response. This response is known as hyperacute rejection and leads to rapid destruction of the transplanted tissue. Deletion of the gene for alpha(1,3)galactosyl transferase, which synthesizes the offending galactose, could eliminate a major target of the hyperacute rejection response and thus lead to the development of animals that can better serve as human tissue donors.

Prions, which arise due to a conformational change in the normal prion protein, are novel infectious agents that cause spongiform encephalopathies in humans and animals. Recently, an epidemic of the neurodegenerative disease, bovine spongiform encephalopathy (mad cow disease) has devastated the British beef industry. Fears have been raised that the infectious agent has crossed species lines and infected humans, causing a form of the neurodegen-erative Creutzfeld-Jacob disease. The unusual resistance of the infectious agent to normal sterilization procedures has raised concerns about contamination of surgical instruments. Thus deletion of the PrP gene in animals would be expected to result in a population of prion-resistant animals. In support of this hypothesis, mice lacking both copies of the PrP genes have been shown to be resistant to infection with scrapie prions.

To generate sheep lacking the alpha(1,3)galactosyl transferase or the prion protein genes, the Roslin group knocked out these genes in ovine fetal fibroblasts. Approximately one to 10 out of a million transfected cells contained the gene targeting event. Selected cell lines with the desired genetic modification were first cultured in low serum medium and then fused to enucleated oocytes. A total of 120 reconstructed embryos were transferred to 78 recipient ewes, resulting in 39 pregnancies at day 35. The two oldest GGTA1 targeted fetuses died in utero at 118 and 130 days of gestation. Of the eight pregnancies that were maintained to term (148 days), four live births were derived from nuclear transfer of PrP targeted cells. Three of these PrP knockout lambs died soon after birth. The remaining lamb had to be euthanized at 12 days of age due to pulmonary hypertension and right side heart failure, which are common problems associated with cloned sheep. Autopsies of the other lambs indicated a common pattern of abnormalities such as distention of the liver, which is suggestive of cardiac insufficiency and kidney displasia. All of these abnormalities have been previously reported for other animals cloned by nuclear transfer.

The high incidence of mortality was unexpected. Because only one of the two functional copies of the GGTA1 or PrP genes was disrupted, no deleterious effect was anticipated. These adverse results suggest that the long term and stringent culture conditions required for selecting cells with a gene targeted event may compromise the ability of the cells to produce viable clones or may be detrimental to proper development of the fetus. The gene targeting event per se, however, is not detrimental because researchers at PPL Therapeutics (UK) have previously shown that sheep containing a gene targeting event at the alpha1 procollagen locus are viable (see " Gene Expression on Target in Sheep," ISB News Report, August 2000).

Although the death of all of the gene-targeted lambs was a disappointment, this report still was able to show that gene targeting could be accomplished at two additional genetic loci. Clearly improvements in the technique for generating live born lambs after gene targeting and nuclear transfer are required; however, this report is a cautiously optimistic first step in being able to precisely modify the genome of livestock species.

Source

Denning et al. 2001. Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep. Nature Biotechnology 19: 559-562.

Eric A. Wong
Department of Animal andPoultry Sciences
Virginia Tech
ewong@vt.edu



SEEDS OF A CANADIAN JUDICIAL CONFLICT: AN UPDATE

On the 29th of March 2001, the Federal Court of Canada ruled that on the balance of probabilities, the defendants in the case of Monsanto v. Schmeiser Enterprises infringed a number of rights under the Plaintiff's Canadian patent number 1,313,830 for Roundup Ready canola. (The patent was issued February 23, 1993, and is due to end in February 2010.) These rights were infringed by the planting of canola fields by Percy Schmeiser in 1998, without leave or license by the plaintiffs, with seed saved from the 1997 crop in which the seed was known, or ought to have been known, by the defendants to be Roundup tolerant and when tested was found to contain the gene and cells claimed under the plaintiff's patent. By selling the product of the seed harvested in 1998, the defendants further violated the plaintiff's patent. Schmeiser Enterprises was ordered to pay $19,832.00 Cnd, representing the profit made from the 1998 canola crop.

However, as of June 19th, 2001, an appeal has been lodged with the Federal Court of Appeal as the defense feels the case warrants rehearing.

Who is Percy Schmeiser
According to legal documents, Percy Schmeiser has farmed in the region of Bruno, Saskatchewan (Canada) where he has lived for more than 50 years. He has grown canola since the 1950s. "He also has an extensive history in municipal and provincial politics, and as a businessman and an adventurer." Schmeiser's Web page, <
http://www.percyschmeiser.com>, outlines much of the legal case and the extensive world travels on which he has embarked as a result of the case.

The Grounds of Appeal
The appeal lodged proposes that Justice McKay was incorrect on the following grounds:

• Patent particulars
Schmeiser's legal team suggest that, in their opinion, Justice McKay erred in determining that a farmer whose field has canola seeds or plants that possess the genetic modification outlined in Patent 1,313,830 has no right to grow, cultivate, harvest, or sell any such seeds or plants, regardless of whether they inadvertently found their way into the field by adventitious means.

They also state the Judge was incorrect in what they believe was the determination that a farmer, who knows or ought to know that there are such GM canola seeds or plants in his/her crop, will infringe the patent on such a crop if he saves and reuses canola seed derived from that crop.

Legal council for Schmeiser holds the belief that the Judge failed to recognize that, in their opinion, a farmer must use or take advantage of the patented gene by in-crop spraying with a glyphosate-based herbicide such as Roundup in order to infringe the patent on the crop.

They also propose that the Judge was wrong not to determine that Monsanto had waived their patent rights by "unleashing" an "invention" into the environment that it cannot control.

Evidence decisions
In the appeal documents, Schmeiser's representatives disagree with the Judge's finding of "no evidence" that the canola seed planted by Percy Schmeiser in 1997 included seed from a field that had swaths and pollen carried into it from a neighbor's Roundup Ready canola field.

They also argue that the Judge gave very little weight to the fact that Monsanto has withdrawn the allegation that Percy Schmeiser had "obtained" canola seed from one or more of their licensed users.

In addition to this, Schmeiser's lawyers insist that, even though Justice McKay found it did not matter how Schmeiser came into the possession of the patented seed, the Judge was wrong to put the onus onto Schmeiser to prove how the seed found its way onto his land, whether by contamination or otherwise.

Testing
Six of the seventeen grounds of appeal submitted on behalf of Percy Schmeiser challenge the actual testing of crops on his land. These include assertions that the Judge gave undue weight and significance to the internal sampling and testing of the canola by Monsanto and correspondingly insufficient weight to the independent testing done on Percy Schmeiser's behalf.

Schmeiser's legal team also disagree with the finding by the Judge that the samples of the 1998 canola crops were properly representative of the fields in question, as there was no expert testimony to support such a finding.

Furthermore, they suggest that the samples from the 1998 canola crop were spoiled and subject to improper tampering by Monsanto. They strongly believe that such tests should be dropped as evidence, because the samples were improperly and/or illegally obtained, which constituted a possible breach in the correct application of the Charter of Rights and Freedoms.

• Money, Money, Money….
Schmeiser's team also challenge the ruling that Monsanto, when awarded damages, was entitled to all the profits made by Schmeiser on his 1998 canola crop, considering the fact that there was no actual finding of the degree or extent to which his crop contained the GM Roundup Ready canola plant.

In addition to this, they are of the opinion that the Judge was wrong to find that Monsanto was entitled to the entire net profit of Schmeiser's 1998 canola crop without proving that the gene that Monsanto claims was present actually conferred any added commercial value to the crop.

Finally
The last ground for appeal argues that Justice McKay was wrong to issue an injunction that Schmeiser's legal team claim impairs Percy Schmeiser from carrying out the traditional farming practice of saving and reusing canola seed during the term of the patent.

Source

Notice of Appeal, Canada Federal Court of Appeal. Schmeiser Enterprises Ltd. v. Monsanto. Court File No. A-367-C1, registered on the 19th of June, 2001. http://www.percyschmeiser.com /appeal.pdf

Shane Morris and Ben Chapman
Centre for Safe Food
University of Guelph
morris@uoguelph.ca



More meetings can be found at
http://www.isb.vt.edu

A Plant Breeding Odyssey
September 10 - 14, 2001
Edinburgh, Scotland, UK

The XVIth Congress of Eucarpia will underline the substantial contribution that Plant Breeding and associated scientific disciplines will make to improving crop production in a sustainable way in the 21st century, and covers every aspect of the science and technology that underpins the multi-disciplinary and multi-commodity nature of plant breeding. It has been deliberately structured to encourage discussion and interaction between delegates.

Themes and Keynote speakers:
• Molecular markers, genomics and bioinformatics
• Transgenics and new crops
• Genetic Resources and Biodiversity
• Breeding for Sustainability
• Quality, Nutrition and Human Health

Contact: CHARISMA
Email: events@charm.co.uk
http://www.eucarpia.org/Congress%202001.htm



Cassava, An Ancient Crop for Modern Times: Food, Health, and Culture
Fifth International Scientific Meeting of the Cassava Biotechnology Network
November 4 - 9, 2001
St. Louis, Missouri

The conference will be devoted to the presentation of cassava as a crop, constraints that limit productivity, and the status of cassava biotechnology. Introductory speakers are: Roger Beachy, The Danforth Center and the Developing World; Missouri Senator Kit Bond (invited): Politics, Science, and Aid; Peter Raven: Conservation and Sustainability; Marc Van Montagu: Biotechnology for Tropical Crops; and Keynote Speaker, Gordon Conway, President, Rockefeller Foundation: Science, Food Crops, and Development.

The program will consist of keynote addresses, general lectures, specialized seminars, and poster sessions. A significant part of these sessions will be reserved for selected oral communications. In addition, there will be evening round tables to review progress and problems.

Contact: Bernadette Delannay
Telephone: 314-516-4583
Fax: 314-516-4582
Email: cbn-v@danforthcenter.org
http://www.danforthcenter.org/iltab/cassavanet/cbnv/



International Conference on Agricultural Science and Technology (ICAST)
November 7 - 9, 2001
Beijing, China

Sponsored by the Chinese Government and cosponsored by UNESCO, World Bank, UNDP, and FAO, this conference will provide an opportunity to sum up the achievements and lessons learned in the 20th century, to exchange policies and experience in the development of agricultural science and technology in different countries, and to envision our joint mission of the development in the new century, through innovation and cooperation on agricultural science and technology around the world. The conference is an international gathering, which covers almost all disciplines of agricultural science and technology. It also includes a Governmental Forum and an Agricultural Business Forum.

Session Topics include:
• Governmental forum on agricultural science and technology
• Sustainable agriculture
• Agbiotechnology
• Post harvest management
• Information technology
• Resources and environment
• Agricultural Business Forum

Contact: ICAST
Email: icast@agscience2001.org
Telephone: +86-10-68511837
Fax: +86-10-68571255
http://www.agscience2001.org/




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