INFORMATION SYSTEMS FOR BIOTECHNOLOGY - NATIONAL BIOLOGICAL IMPACT ASSESSMENT PROGRAM
ISB News Report
June 1998

NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY


IN THIS ISSUE:
Patenting Life In Europe
Field Testers Taken To Task
Regulatory Gene Confers Resistance To Multiple Diseases
Chitinase-Expressing Tobacco: Bad News For Budworms
Cassava Dreams Spurred By First Molecular Genetic Map
Human Clinical Trials Show Effectiveness Of Transgenic Plant-Derived Pharmaceuticals
Seedless Tomatoes On The Way
Transposon-Mediated Transformation Of Mosquitoes
Major Moves: An Acquisition Update

News and Notes


Patenting Life in Europe

On 12 May 1998 the European Parliament approved the Biotechnology Patent Directive after ten years of discussion. One of the major rate-limiting steps of the Biotechnology Revolution has been the legal issue of prohibiting worldwide patenting of transgenic inventions. This vote removes legal prohibitions to patenting transgenics.

The following is summarized from a patent commentary by Breffni Baggot in the March 1998 issue of Nature Biotechnology. Two laws have blocked the patentability of transgenics: the European Patent Convention's (EPC's) Article 53(a) blocks patentability of inventions whose commercial use would be contrary to public policy and Article 53(b) excludes "plant and animal varieties" from patentability.

Interpretations vary from country to country and therein lies the rub. The European Patent Organization (EPO) and EU member states have selectively issued or rejected patents. Two cases have made the interpretation especially difficult, the granting of a patent to the "Harvard" mouse and the rejection of a Plant Genetics Systems transgenic plant patent.

Discontent over the inability to explain this discrepancy led to the drafting of this new legislation known as the EU Biotechnology Patent Directive, which is more favorable toward patenting transgenics and less subject to idiosyncratic interpretation. The Directive redefines EPC Article 53 to avoid the result in the Plant Genetics Systems case and defines both patentable plants and animals and plant and animal varieties.

If sorting out this legal process seems burdensome, similar things happened in the computer industry. The computer software industry ultimately claimed a mathematical algorithm in conjunction with a physical process to override the U.S. Supreme Court's rejection of patenting an algorithm alone. The biotechnology industry would do well to demonstrate the industrial application of transgenic animal and plant materials.

More information can be found on the following web sites:

1. Notice of the decision, Europe OKs Biotech Patents, http://www.bric.postech.ac.kr/science/97now/98_5now/980512c.html

2. Final text of the EC Directive on the legal protection of biotechnological inventions, http://www.wuesthoff.de/c.htm

3. Commentary on the EC Directive, http://www.wuesthoff.de/e.htm, http://ci.mond.org/9506/950613.html

4. A non-technical summary of intellectual property rights issues in agricultural biotechnology, the ISB News Report Special Issue On Intellectual Property Rights, http://www.isb.vt.edu/news/1995/news95.may.html

Edward Weck
BioTec Innovation
Northfield, MN
eweck@microassist.com


Field Testers Taken to Task

Leading biotechnology companies that failed to stick to agreed plans for field experiments with genetically engineered plants have had their knuckles rapped by Britain's gene police. Though not fined, the culprits have been named publicly and some have been forced to rip up the plots.

The Advisory Committee on Releases to the Environment (ACRE), which advises the British government on the safety of releasing genetically engineered organisms, last week publicized a rogues' gallery of companies and research labs that have breached the terms of their approvals to carry out field experiments. The information was reported in the April 4, 1998 issue of New Scientist.

The companies are Monsanto of St. Louis, Missouri, the world's largest agricultural biotech firm; Plant Genetic Systems of Ghent, Belgium; AgrEvo of Frankfurt, Germany; and Nickerson Biochem of Cambridge, England. The other miscreants are the Scottish Crop Research Institute near Dundee and the National Institute of Agricultural Botany in Cambridge, England (see table below). Jony Beringer, a microbiologist at the University of Bristol who chairs ACRE, says "This should help to show that the government is not acting like a doormat," adding "I'm all for naming and shaming, as this is worth many times more than fines."

Anyone planning to grow an experimental plot of genetically engineered plants in Britain must first put detailed proposals to ACRE. The breaches highlighted by ACRE were uncovered by the Health and Safety Executive. Nickerson Biochem and Monsanto both allowed crops to grow much nearer to unmodified plants than agreed. The two companies said that the errors were due to misunderstandings by the contractors who carried out the trials, but they accept responsibility.

"It's completely inexcusable, and we have done everything we can to avoid it happening again," says Tina Barby, Director of Nickerson Biochem.

P. Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise
Hyderabad, India
ipe@hyd.ap.nic.in

Biotech Company Crop Breach of Contract
Monsanto Herbicide resistant oilseed rape Buffer zone surrounding crop too small
Plant Genetic Systems Herbicide resistant oilseed rape with male sterility Failed to notify conservation officials or public about trials
AgrEvo Herbicide resistant wheat Failed to implement measures to limit escape of pollen
Nickerson Biochem Oilseed rape with extra phytase gene Buffer zone surrounding crop too small
Scottish Crop Research Institute Potato resistant to leaf roll virus Surrounded plants with beans rather than cereals
National Institute of Agricultural Botany Herbicide resistant oilseed rape Seed scattered outsidedesignated area

Plant Research News


Regulatory Gene Confers Resistance to Multiple Diseases

Researchers at Duke University have identified a gene that triggers what is called the acquired resistance response in plants by turning on a battery of pathogenesis-related genes. Overexpression of this gene beefed up the plant's defense response against both fungal and bacterial pathogens.

The report appears in the latest issue of PNAS. The strategy is similar to that employed to enhance the plant tolerance against cold by turning on a regulatory gene (see May 1998 ISB News Report).

In the face of pathogen attack, plants generally can have two types of defense reaction. One is a hypersensitive response that restricts the spread of specific pathogens at the site of infection. It is an "all or none" response involving a single plant R gene corresponding to a specific avirulence gene in the pathogen, the so called gene-for-gene interaction. The other type of response is called systemic acquired resistance (SAR), a non-specific reaction involving expression of many pathogenesis-related (PR) genes.

Whereas R genes provide a high level of resistance against specific pathogen races, they are vulnerable to pathogen evolution and disease resistant varieties become susceptible quickly. On the other hand, PR genes, which individually do not provide adequate protection, work collectively to provide a modest but long term resistance against many pathogens. Both gene types have been cloned recently from many plants and are the subject of intense molecular scrutiny.

The Duke team led by Xinnian Dong cloned a "master-switch" gene (NPR1) in Arabidopsis that regulates many downstream PR genes. NPR1 expression is enhanced by pathogen attack or chemicals which are known to trigger SAR such as salicylic acid. The NPR1 gene is involved in the transduction of the salicylic acid signal leading to SAR. Researchers introduced a copy of this gene under the control of the constitutive CaMV35S promoter back into Arabidopsis.

Transgenic plants having only a modest increase in NPR1 protein showed dramatic resistance not only to the bacterial pathogen Pseudomonas syringae but also to the fungal pathogen Pernospora parasitica. Just three days after infection, the growth of bacteria was inhibited more than a thousand-fold in transgenic plants. These lines also showed an increased transcription of many PR genes, suggesting that the NPR1 gene activates a range of defense-related genes which may act synergistically to confer disease resistance.

Overproducing the NPR1 protein resulted in "stronger rather than quicker" induction of PR genes. The reaction was similar to the disease resistance response seen in wild type plants when sprayed with chemicals that trigger SAR. Interestingly, one transgenic line producing negligible amounts of NPR1 protein (possibly due to cosupression) remained highly susceptible to both pathogens and showed no increase in PR gene expression.

The Duke study hints at a tantalizing possibility that crop plants with durable and broad-spectrum resistance against many destructive diseases can be developed using just one gene. Several crops, including potato, corn, wheat, cabbage and canola, contain DNA sequences similar to the NPR1 gene, and Dong has already cloned the gene from tobacco and tomato. She believes that NPR1-mediated resistance will be more durable than R genes, as the pathogen will have the insurmountable task of overcoming many activated plant defense genes in order to become virulent. Further, as the NPR1 protein is activated only under disease attack it may not drain the plant's resources and may be less likely to impose strong selection pressure on the pathogen. Dong's group is now testing the transgenic Arabidopsis against other diseases.

Reference

Cao, H., X. Li, and X. Dong. 1998. Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc. Nat. Acad. Sci. USA 95:6531-6536.

C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@tusk.edu


Chitinase-Expressing Tobacco: Bad News for Budworms

The ability to introduce foreign genes into plants has allowed the development of a variety of strategies for improving resistance to insect predation and diseases of crop plants. One such strategy has been to deploy a gene that was cloned from a pathogen or pest for biological control. Expression of this gene at an appropriate time and level disrupts the development of the same or a related pathogen or pest. Examples include expression of viral coat protein genes to confer resistance to virus infection, and, more recently, use of insect chitinase genes to interfere with the growth and feeding of insect pests.

Expression of chitinase in the insect gut normally occurs only during molting, when the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected by an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing cDNA encoding a tobacco hornworm (Manduca sexta) chitinase into tobacco via Agrobacterium-mediated transformation.

A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced in budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. This result illustrates that an insect chitinase transgene can enhance plant resistance to budworm neonates feeding on tobacco.

In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenic plants. Why hornworm larvae were not affected is unclear. One possible explanation is that the concentration of the enzyme was too low to negate inactivating mechanisms likely present in this homologous system, given that the specific activity of the truncated form is about one-fourth that of the full-length enzyme.

Interestingly, however, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sub-lethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on control plants having only the Bt treatment. Foliar damage was also reduced. For tobacco hornworm, the effect was seen on transgenic plants having a foliar coating of 288 ng of Bt toxin per gram fresh weight.

A similar Bt toxin-chitinase transgene interaction was observed with the tobacco budworm. Chitinase expressing plants coated with 360 ng of toxin per gram fresh weight exhibited a 56% reduction in their mean foliar damage index relative to nontransgenic controls, and the biomass of surviving larvae was reduced by 89%.

This observation suggests the Manduca chitinase gene could also be useful for potentiating the effectiveness or increasing the spectrum of Bt insect control proteins. It may have utility as a companion transgene, which, when engineered into a plant together with a Bt gene, enhances the effectiveness of the Bt gene. Alternatively, a crop cultivar expressing a chitinase transgene might be used to potentiate the action of a foliar Bt application. This approach would provide an alternative to full-season exposure to transgene encoded Bt toxin, which raises a management issue regarding the development of insect resistance.

Further exploration of the potential for insect control by the Manduca or other chitinases, alone or in combination with other genes for insecticidal proteins, is warranted.

Reference

Ding, X. et al. 1998. Insect resistance of transgenic tobacco expressing an insect chitinase gene. Transgenic Research 7:77-84.

P. Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise
Hyderabad, India
ipe@hyd.ap.nic.in


Cassava Dreams Spurred By First Molecular Genetic Map

Nigerian plant geneticist Martin Fregene has a dream: to find the genes that control agronomic traits in cassava. This knowledge could be used to enhance cassava's traditional role as provider of food security in Africa, and future role as an industrial crop world-wide. The quest has spurred him to develop a molecular genetic map for cassava, the first such map for a major food crop generated outside of an industrialized country.

Fregene and his colleagues developed the map at the International Center for Tropical Agriculture (CIAT) near Cali, Colombia, and reported it in the August 1997 issue of Theoretical and Applied Genetics. Fregene is convinced the effort will help brighten the picture for what he has called "the genetically least understood of any of the staple crops that feed mankind, including rice, maize, wheat, and potatoes." Indeed, over 500 million people depend on cassava for their food and livelihood in the developing world, with 50 million consuming it twice daily in Fregene's home country of Nigeria.

Cassava biotechnology news last appeared in the ISB News Report in July and August, 1996. An editorial by Indra K. Vasil in the June 1996 issue of Nature Biotechnology, claiming that "the technology to improve cassava ... is a product of developed countries," raised questions regarding the appropriate use of, and access to, such technology. These remarks were countered by Ann Marie Thro's detailed description of the Cassava Biotechnology Network (CBN), two-thirds of whose 300 member researchers work in cassava-growing countries, i.e. the developing world.

Nearly two years later, it is clear that much groundbreaking cassava biotechnology research is being done in the southern hemisphere. A soon-to-be-published book (1) describes a number of important advances in the areas of molecular genetics, genetic transformation and regeneration, and micropropagation.

The molecular genetic map of cassava is expected to push forward the study of the genetics of economically important traits and genetic diversity in cassava. Two good examples, both underway at CIAT, are ongoing quantitative trait loci studies of root quality traits and early bulking, and a recently concluded study of genetic relationships between 600 cassava genotypes and their wild relatives using microsatellite markers that were developed during the mapping project. More recently, genes of known function involved in starch biosynthesis and cyanogenesis, as well as seven expressed sequence tags (ESTs), have been added to the map. Addition of ESTs to the cassava map is only just beginning; it is part of ongoing efforts to saturate the map with highly polymorphic, PCR-based molecular markers.

The last several years have also brought advances in genetic transformation and successful recovery of genetically transformed cassava plants. This was made possible by development of a system of organogenesis using somatic embryos as the source of cotyledon explants. Cells at or close to the cut edges of the explants can be induced to form shoots; thus they are suitable for Agrobacterium-mediated gene transfer. Improved regeneration through this method has been accompanied by enhanced selection methods, including use of the firefly luciferase gene, either alone or in combination with the phosphinoacetyl transferase gene which confers herbicide tolerance.

The above review should provide a useful context in which to evaluate the appearance of the molecular map for cassava. In a recent interview, Fregene discussed the implications of the map and its end users. He pointed out that the consequences of hampered breeding in cassava until now include production not meeting demand in Africa's sub-Saharan region, where over 90% of yield is consumed as food. This has caused an increase in field acreage, mostly into marginal lands, and price increases. What's more, the erratic supply of cassava roots prevents new marketing and post-harvest opportunities, impeding acquisition of much needed skills and income.

The advent of genome studies using genetic maps and molecular markers and plant transgenesis have all provided ways around obstacles inherent to cassava such as its long growth cycle and heterozygous nature. At present efforts are geared to gene tagging of important root quality and earliness traits. Additional work includes localizing and cloning resistance genes to the devastating African cassava mosaic disease and cassava bacterial blight. It is hoped that these efforts provide a solution to frequent disease epidemics, as well as a means of understanding the mechanisms of resistance. The latter is the subject of Fregene's current research (2).

As for the end users of these technologies, Fregene said that genetic engineers, germplasm specialists, and plant breeders are now taking advantage of his team's research results. "Marker technology in general will not reach the farmer quite yet," he pointed out. "This will take three or four more years of research." The geneticist also added that "four years is nothing when you have a dream."

References

1. Thro, A. M. et al. 1998. Genetic biotechnologies and cassava-based development of marginal rural areas. In: T. Hohn and K. Leisinger, eds. Gene- and Biotechnology of Food Crops in Developing Countries. Springer Verlag, New York, Vienna, in press.

2. Fregene, M. et al. 1998. AFLP Analysis of African cassava germplasm resistant to the Cassava Mosaic Disease (CMD). Theoretical and Applied Genetics, in press.

Timothy Pratt
Journalist
Cali, Colombia
thipra@norma.net


Human Clinical Trials Show Effectiveness of Transgenic Plant-Derived Pharmaceuticals

For centuries, plants have been a valuable source of natural pharmaceuticals. In the past decade, intensive research has been focused on expanding the use of plants as pharmaceutical production systems by genetic engineering. It is now clear that plants can be manipulated to produce a wide variety of such compounds, from vaccine antigens and monoclonal antibodies to pharmaceutically valuable secondary metabolites. Two of the seminal papers which gave proof of the concept of the use of transgenic plants as pharmaceutical production systems were published back-to-back in the May 1995 issue of Science. Now, exactly three years later, in the May issue of Nature Medicine the same two groups reported the results of successful human clinical trials with their transgenic plant-derived pharmaceuticals: an edible vaccine against E. coli-induced diarrhea (1) and a secretory monoclonal antibody directed against Streptococcus mutans, for preventative immunotherapy to reduce incidence of dental caries (2).

An Edible Vaccine in Transgenic Potatoes

Research in the Plants and Human Health group at the Boyce Thompson Institute for Plant Research at Cornell University, led by Drs. Charles Arntzen and Hugh Mason, is focused on developing both production and delivery systems for subunit protein vaccines. They have reported that transgenic plants can express a variety of antigenic proteins, such as hepatitis B virus surface antigen, Norwalk virus capsid protein, and the B subunit of the Escherichia coli heat labile enterotoxin (LT-B). In 1995, this group showed that not only could transgenic potato plants express the E. coli LT-B protein, but also that tubers expressing this protein could induce a specific immune response against LT-B when fed to mice as part of their normal diet (3). These results suggested that transgenic plant tissues expressing vaccine antigens could be used for immunization against a myriad of diseases, and raised hopes that this technology might solve many of the problems associated with delivery of safe, effective vaccines to people in developing countries. Production of recombinant subunit vaccines could be as cheap as agriculture, distribution as convenient as marketing fresh produce, and administration as simple and as safe as feeding a baby a banana! The report describing the results of the first human clinical trial of a plant-derived vaccine provides further important proof that transgenic plants could be used as "edible vaccines". This trial used E. coli LT-B-expressing potatoes produced by the Boyce Thompson Institute group.

Enterotoxigenic E. coli (ETEC) and Vibrio cholerae are the primary pathogens responsible for acute watery diarrhea. Both bacteria initiate disease by colonizing the intestinal epithelia and both produce multi-subunit enterotoxins, which cause the diarrheal symptoms. The heat labile enterotoxin (LT) of ETEC is closely related to cholera toxin (CT). There is currently no reliable and effective vaccine against either ETEC or cholera. There is, however, evidence that suggests that oral vaccination against the B-subunit of either toxin can induce production of mucosal antibodies which can neutralize the toxicity of the respective holotoxin by preventing its binding to gut cells.

In the clinical trial, two groups of volunteers consumed either 50 g or 100 g of raw potato tubers expressing LT-B (equivalent to 0.5 mg or 1 mg LT-B per dose, respectively) and were compared with a third group that ate untransformed potato tubers. The first two groups developed specific anti-LT-B mucosal and systemic immune responses while the control group did not. These responses are comparable to those observed when humans are challenged with 109 ETEC bacteria. The human clinical trials demonstrate that edible plant vaccines are immunogenic in humans, as was previously shown in mice; proving that they can protect humans against a challenge is the next logical step.

A Plant-Derived Recombinant Antibody

In 1995, Julian Ma of Guy's Hospital in London, UK, and colleagues showed that transgenic tobacco plants could express and assemble recombinant secretory antibodies. This was in itself a significant advance since these molecules are fairly complex, consisting of four separate polypeptide chains. Ma's group expressed a hybrid secretory monoclonal antibody (SIgA/G) directed against the cell surface adhesion protein of S. mutans. When the antibody is present in the oral cavity it prevents bacterial colonization and subsequent development of dental caries. The authors expressed each of the four proteins in separate tobacco lines, and created a line which expressed all four by sexually crossing the four lines (4). They were able to show that the four polypeptides-- a hybrid IgG-IgA heavy chain, IgG light chain, a joining chain, and a secretory component-- assembled into a functional secretory immunoglobulin molecule.

In their recent paper, Ma's group compared the transgenic recombinant SIgA/G molecule with its parental mouse IgG monoclonal antibody. As expected, the plant-derived molecule and the parental antibody had similar affinity constants; the plant molecule had higher functional affinity since it is a dimeric structure, with four antigen binding sites, in comparison with the monomeric IgG molecule. For the human trial, the oral cavity of adult human volunteers was effectively sterilized with a bacteriocidal mouthwash, and the antibody solution applied to the teeth. Overall, the results of this trial were spectacular. The plant-derived SIgA/G survived for longer periods of time than the IgG (3 days compared with 1 day) in the human mouth, probably because SIgA molecules have markedly enhanced stability in comparison with IgG. The recombinant antibody prevented recolonization of the teeth by S. mutans, but not other bacteria, for at least four months, while volunteers who received control treatment all had significant S. mutans recolonization within two months.

In both human clinical trial studies, there were no major side-effects observed. These reports therefore represent significant advances in plant biotechnology, and have shown that transgenic plants may indeed be cost-effective, efficient and effective production systems for protein pharmaceuticals.

References

1. Tacket, C.O., H.S. Mason, G. Losonsky, J.D. Clements, M.M. Levine, and C.J. Arntzen. 1998. Immunogenicity of a recombinant bacterial antigen delivered in a transgenic potato. Nature Medicine 4: 607-609.

2. Ma, J.K-C., B.Y. Himat, K. Wycoff, N.D. Vine, D. Chargelegue, L. Yu, M.B. Hein, and T. Lehner. 1998. Characterization of a recombinant plant monoclonal secretory antibody and preventative immunotherapy in humans. Nature Medicine 4: 601-606.

3. Haq, T., H.S. Mason, J.D. Clements, and C.J. Arntzen. 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268: 714-716.

4. Ma, J.K-C., A. Hiatt, M. Hein, N.D. Vine, F. Wang, P. Stabila, C. van Dolleweerd, K. Mostov, and T. Lehner. 1995. Generation and assembly of secretory antibodies in plants. Science 268:716-719.

Kenneth E. Palmer
Boyce Thompson Institute for Plant Research
Cornell University
kep12@cornell.edu


Seedless Tomatoes on the Way

In the wake of the recent development of seedless eggplant by an Italian research team, Yi Li, a Kansas State University scientist, now reports the development of a seedless tomato. The two studies used a similar approach in which seedlessness was achieved by genetically engineering parthenocarpy through overproduction of hormones necessary for fruit development (see the February 1998 ISB News Report).

While the small seeds in tomato are not bothersome for consumers, the ability to produce tomato fruits without seeds may afford considerable advantages for the grower. Normally, pollination and subsequent seed production are required for good fruit development in most plants. Seeds in developing fruit trigger the synthesis of auxins and cytokinins, plant hormones that promote cell division and expansion. Bad weather during flowering can interfere with pollination, resulting in a bad tomato harvest. Thus genetic modification of the crop to reduce its dependency on pollination for fruit development could reduce the constraints of weather, insect pollinators, and growing season.

Li developed seedless tomatoes by expressing two Agrobacterium phytohormone genes under the regulatory control of promoters that are specifically expressed in developing ovaries and fruit. An unexpected bonus in the engineered tomatoes was the high solids content seen in the fruits. Li suspects that the seedless tomato may funnel its resources towards higher solids that would otherwise have gone into producing seeds and the jelly-like substance surrounding them.

Both seedlessness and high solids are valuable traits for the tomato processing industry, where most of the tomato harvest ends up. Processors routinely remove the seeds and reduce the water content of tomatoes before cooking up ketchup or pasta sauce.

Currently, the tomatoes need to be emasculated (pollen sacs removed) to obtain completely seedless fruits, a laborious process. A Dutch biotechnology company, which is field testing the transgenic tomatoes, is working to introduce a female sterility gene into these lines. Li and the company are also developing seedless watermelons through genetic engineering, as they believe that this would result in better tasting fruits with extended shelf life.

Reference

Kansas State University News Release, April 21, 1998. http://www.newss.ksu.edu/WEB/News/NewsReleases/seedless4218.html

C.S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@tusk.edu

Animal Research News


Transposon-Mediated Transformation of Mosquitoes

Mosquitoes serve as a vector for transmission of a number of pathogens such as yellow fever virus, dengue virus and the malarial parasite. Attempts to develop pathogen-resistant mosquitoes have been hampered by a lack of a reliable method to stably transfer foreign DNA into the mosquito germline. This obstacle has finally been overcome. In the March 1998 issue of the Proceedings of the National Academy of Sciences (USA), researchers at the University of California, Irvine and the Centers for Disease Control in Atlanta report methods for the stable transfer of foreign genes into the genome of the mosquito Aedes aegypti.

Borrowing from the molecular tools developed for other insect species, the fly transposable elements Hermes and mariner were modified for use in mosquitoes. Transposable elements or transposons are small DNA fragments that can move around the genome of the host by a process of replication/excision and integration. Thus when a transposon that has been modified to contain foreign DNA integrates into the host genome, the foreign gene is also cointegrated.

To readily monitor the fate of the foreign DNA, an eye color gene, again borrowed from flies, was cloned into a transposon residing in a plasmid. This construct was coinjected into mosquito embryos along with a plasmid encoding the enzyme transposase, which is necessary for the excision of the transposon DNA from the plasmid. A mutant strain of white-eyed mosquitoes was used as a recipient. Transformed white-eyed mosquitoes which express the new eye color gene developed colored eyes. Using this phenotypic trait as a marker, the foreign gene was observed to be transmitted through the germline according to Mendelian rules and to be stable for at least ten generations.

The development of these transformation systems opens up the real possibility of genetically engineering pathogen-resistant mosquitoes. For example, Aedes mosquitoes could be made resistant to yellow fever virus or Dengue virus and Anopheles mosquitoes, an important malaria vector, could be genetically manipulated to control outbreaks of malaria. Mosquitoes also serve as a vector for the transfer of the Eastern equine encephalitis (EEE) virus from wild birds to humans and horses. EEE virus attacks the central nervous system and infection is almost always fatal in horses. An EEE virus resistant mosquito could greatly reduce the incidence of this fatal disease in horses.

Development of transgenic mosquitoes is just the first step towards the implementation of a biological control program. Further research is needed to identify genes that confer resistance to pathogen infection. The appropriate strategy for the safe and effective spread of the recombinant gene to wild mosquito populations needs to be evaluated. In addition, the risks associated with releasing transgenic mosquitoes into target areas must be assessed. Although a number of questions still remain to be answered, the light in the distance for insect biotechnologists is drawing nearer and nearer.

References

1. Jasinskiene, N. et al. 1998. Stable transformation of the yellow fever mosquito, Aedes aegypti, with the Hermes element from the housefly. Proc. Natl. Acad. Sci. USA 95:3743-3747.

2. Coates C.J. et al. 1998. Mariner transposition and transformation of the yellow fever mosquito, Aedes aegypti. Proc. Natl. Acad. Sci. USA 95:3748-3751. Eric Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu

Industry News


Major Moves: An Acquisition Update

It seems to get harder and harder to write about the business side of agricultural biotechnology without discussing Monsanto in one way or another. This month is no exception. In fact, in a flurry of recent activity, Monsanto has further strengthened its position in the crop biotechnology arena. There is also some talk that the hunter might also be the hunted, as recent divestitures on the part of chemical giant DuPont have led to speculation that Monsanto might become an acquisition target of the Wilmington, Delaware (USA)-based company.

In mid-May, Monsanto announced that it had reached agreements with DEKALB Genetics and Delta & Pine Land Company to acquire the two companies (1). DEKALB is a global leader in agricultural genetics and a top hybrid seed corn company, while Delta & Pine is a major breeder, producer, and marketer of cotton seed. These acquisitions by Monsanto allow the company to "provide both technology and global reach by creating broader seed platforms that enable [Monsanto] to better connect traits to the needs of growers and processors, and allow [Monsanto] to more quickly anticipate new markets or marketplace trends."

Monsanto agreed to ante up a total of $4.2 billion in cash and stock for the two seed companies, paying over $100 per share for DEKALB ($2.3 billion) for the 60% of the company it did not already own, and about $1.9 billion for Delta & Pine (2). The price that Monsanto agreed to pay for DEKALB is nearly three times the price that the shares traded for in February prior to the Roberts' Family (founders and holders of a controlling stake) decision to put its shares up for sale. The premiums reflect Monsanto's desire to continue building its crop biotechnology and life sciences business.

There was some concern raised in a recent Wall Street Journal article about Monsanto's debt level given the magnitude of its acquisition activities in the past few years (2). In the same article, Monsanto CEO Robert Shapiro was quick to note that this would not be a problem as Monsanto operates with significant cash flow and is capable of paying down debt very quickly. There was also concern as to the ability of Monsanto to carry out, on its own, its aggressive agbiotech vision. This has led some to speculate that Monsanto will ultimately seek to align itself with a larger partner to create the necessary critical mass to be a major player in the biotechnology-driven agricultural world of tomorrow.

One such possible partner, DuPont Co., recently shed its Conoco oil unit, generating an estimated $25 billion which the company has stated it will likely use to invest in its life sciences business (3). The sale of the Conoco unit is thought by some to precede a major acquisition in the life sciences area by DuPont. Monsanto has been mentioned as one possible target, along with Zeneca and Pioneer Hi-Bred (of which DuPont already owns 20%). DuPont's expectations for life sciences and biotechnology are not paltry. The company anticipates that as much as a third of earnings will come from this area in another five years, compared with only about 18% now. DuPont has been working in recent years to establish its life sciences presence, having last year spent $1.7 billion for its 20% stake in Pioneer Hi-Bred.

Given the feverish pace at which acquisitions have been taking place in the last couple years, it is not unreasonable to expect some kind of action on the part of DuPont in the near future. What exactly that will be is yet to be determined, and the rumors are flying. At the end of May, there was a report that DuPont might purchase healthcare biotechnology giant Amgen (4). Although neither company discussed the proposition publicly, there were mixed sentiments among Wall Street analysts as the validity of the rumors. As always, stay tuned...

References

1. Monsanto Acquires Two Seed Companies To Broaden Availability Of Agricultural Biotechnology. Press Release (http://www.monsanto.com), May 1998.

2. Kilman, S. Monsanto Buys Two Companies for $4.2 Billion. The Wall Street Journal, May 12, 1998.

3. Warren, S. DuPont Plans to Shed Conoco Oil Unit. The Wall Street Journal, May 12, 1998.

4. Seachrist, L. Amgen Stock Jumps On Rumor DuPont May Buy Out Company. BioWorld Today, Vol. 9, No. 99, May 26, 1998. William O. Bullock
Institute for Biotechnology Information,
LLC Research Triangle Park, NC
http://www.biotechinfo.com


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2. To have the News Report automatically emailed to you, send an email message to news@nbiap.biochem.vt.edu and type subscribe newsreport [your_name] in the message section. (Your name is not used in any way; it is simply required by the software).

ISB News Report
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Blacksburg, VA 24061-0308