INFORMATION SYSTEMS FOR BIOTECHNOLOGY - NATIONAL BIOLOGICAL IMPACT ASSESSMENT PROGRAM
ISB News Report
October 1997

NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY

IN THIS ISSUE

Agricultural Biotechnology International Conference '98
Update on the ISB Website
Sunflower Gene Improves Grain Legume Protein
Engineering Plants to Manage Stress
How Now Green Cow?
Vaccinating Animals to Control Insect Pests
Novartis and Chiron Form Alliance for Combinatorial Chemistry


NEWS AND NOTES

AGRICULTURAL BIOTECHNOLOGY INTERNATIONAL CONFERENCE '98
Mark your calendars now for ABIC '98, the agricultural biotechnology international conference to be held June 9-12, 1998 in Saskatoon, SK Canada. The conference is sponsored by Saskatchewan Agriculture and Food, Ag-West Biotech Inc. and a team of 21 corporate and institutional sponsors. The ABIC '98 theme, "Agbiotech: the Science of Success," will focus on strategies for the commercialization of agbiotech products.

ABIC '98 will feature tours of Saskatoon's agbiotech industry sites, 55 expert presentations by 40-plus speakers, poster presentations and a trade show. Presentation topics include:

While the overriding theme is commercial applications of agricultural biotechnology, the program contains enough depth for delegates interested in delving into the details of business applications, bringing products to market and venture financing.

Plenary Speakers
A number of plenary speakers have already confirmed their participation. Dr. Anatol Krattiger, Executive Director of the International Service for the Acquisition of Agri-Biotech Applications Americentre (ISAAA) at Cornell University will address the Importance of Agbiotech to Global Prosperity. Dr. Richard Gill, Senior Vice-President and General Manager of BTG USA Inc. will share case studies of commercial applications for biotechnology in his presentation of Emerging Market Opportunities. Nobel Prize winner Dr. Michael Smith of the University of British Columbia, will discuss Lessons from the First Stages of Human Genome Sequencing.

Other speakers include Dr. Robert Cooper of McMaster University, Mr. Andrew Dickson, Secretary General of EuropaBio in Belgium and Dr. Yutaka Tabei, Deputy Director of the Innovative Technology Division of the Research Council Secretariat, Japanese Ministry of Agriculture, Forestry and Fisheries.

Conference Streams
More than 30 sessions are organized into four main topic streams: Plant and Crop Development, Animal Science, Microbial Science, and Commercial Development. A partial list of speakers includes:

Plant and Crop Development

Animal Science

Microbial Science

Commercial Development

Posters
Participants are invited to present research findings at the conference Poster Session. Research is welcomed from all areas of agricultural biotechnology including new developments from industry. Abstracts will be published in the Conference Highlights for ABIC '98. Application forms and guidelines for abstract submission and poster presentation are available from the ABIC '98 Secretariat. Since the number of posters will be limited by available space, submissions will be considered on a first-come, first-served basis.

For more information contact the ABIC 98 Secretariat c/o The Signature Group, 608 Duchess Street, Saskatoon, Saskatchewan, Canada S7K 0R1. Phone 306-934-1772; fax: 306-664-6615; e-mail siggroup@sk.sympatico.ca or visit their web site at http://www.lights.com/abic/.

UPDATE ON THE ISB WEBSITE

New Risk Assessment Information Available
ISB has put online the Proceedings of Biotechnology Risk Assessment Symposia that were held from 1994 to 1996. The meetings presented risk assessment research funded through the USDA, EPA and Environment Canada. The conference series ended in 1996 as EPA and Environment Canada discontinued their grant programs.

The online version of the Proceedings provides the text of 117 papers presented over the three year period and were converted from the printed version, originally compiled by the University of Maryland Biotechnology Institute. A keyword search "engine" can be utilized to search the entire collection. Work will begin soon to scan and link the graphics, charts and figures accompanying these papers.

We Need Your Help
As you may have discovered (we hope), the ISB website provides a tremendous amount of information for your use, some found nowhere else on the web. Guided by the philosophy that an incomplete set of information is an inaccurate set of information, we welcome your help on a few items. A few of the more difficult lists to maintain are:

If you notice that we are missing items in any of our collections, please email us the missing information or advise us where to get it. As always, our focus is on agricultural and environmental biotechnology. Please feel free to contact us at nbiap@vt.edu with any questions or needs you may have regarding agbiotech information.

Doug King
Information Systems for Biotechnology
nbiap@vt.edu

PLANT RESEARCH NEWS

SUNFLOWER GENE IMPROVES GRAIN LEGUME PROTEIN
A plant-based diet is surely a healthy one, but it compromises on certain key nutrients necessary for our body: essential amino acids. Humans and animals cannot synthesize essential amino acids and thus they need to be supplied in the diet. Most plants are deficient in one or more of these critical protein components while milk, meat or egg contain them in adequate amounts. Grain legumes such as soybean, bean or peanut, which serve as a source of valuable protein in the diets of humans and livestock, are especially deficient in the sulfur-containing amino acids methionine and cysteine. Thus, grain legume feed for pigs and poultry is supplemented with synthetic methionine which these non-ruminant animals can then convert to cysteine. Even ruminants such as sheep benefit from methionine supplements in their food and respond by increasing wool production. Adding a synthetic amino acid to animal feed, however, is an expensive option.

Now, a research group led by Thomas Higgins at the Commonwealth Scientific and Industrial Research Organization (CSIRO) in Canberra, Australia has come up with a biotechnological answer to this problem. They have developed a nutritionally improved legume by transferring a methionine-rich protein gene from sunflower into lupin, an important animal feed legume in Australia (1). Because lupin, like most other legumes, was intractable to regeneration, the CSIRO group first had to develop an improved tissue culture system for this crop. The genetic engineering entailed introducing the sunflower seed 2S albumin (SSA) gene, expressed under the control of a seed-specific promoter from peas. Although the frequency of transformation was low, they obtained a few transgenic plants and selected one line in which the SSA protein constituted 5% of the total protein.

When seven thousand plants from this line were tested in the field, there were no differences in seed yield or total protein levels compared to control plants. Transgenic seeds with the sunflower gene, however, had 95% more methionine but 12% less cysteine than the untransformed control line. The total sulfur in seed meal remained the same between the two lines, but was allocated differently - the transgenic seed meal contained increased amino acid sulfur and a proportionately reduced amount of oxidized sulfur held as sulfates.

The nutritive superiority of the engineered lupin became clear in animal feeding studies. Rats fed on transgenic lupin meal gained 17% in body weight within eight days while those receiving standard lupin food increased their weight by only 3%. The rat study also showed that lupin engineered with the sunflower protein had 23% higher net protein utilization than the control, an indicator of how well protein was digested and its nitrogen absorbed in the body. This value was similar to those obtained with methionine- supplemented lupin meal.

The Australian group rightfully claims that their study is the first report of a "genetically engineered improvement in the nutritive value" of a grain legume crop and an animal feed crop; earlier studies were conducted with the model plant tobacco. Classical plant breeding has had little success in altering the essential amino acid composition of plants, and thus plant molecular biologists have attempted to improve the nutritive value of field bean and soybean through alteration of native protein sequences. But instability of the altered proteins or low expression levels of introduced genes in the plant have curtailed major advances. In a recent well-publicized case, the giant seed company Pioneer Hi-Bred International developed transgenic soybean with a sulfur-rich protein gene from Brazil nut. However, a subsequent study by University of Nebraska scientists showed that the engineered soybeans retained the human allergenicity of Brazil nut, and the company apparently stopped further research in this direction.

The CSIRO group thus chose a protein from sunflower as the seed is not known to cause any allergic reactions; it is also resistant to degradation by microbes in the rumen but readily available in the hindgut of sheep. Now, they are conducting detailed studies on the agronomic performance of the transgenic lupin and testing its nutritive role in sheep, pigs and chickens. The Australian study, if successful, offers a greater promise beyond well-fed livestock. It may also spur similar research on crops of importance in the human diet, where similar successes would go a long way toward alleviating the inadequate nutrition that afflicts people in developing countries where plants are the major source of dietary protein.

Reference

1. Molvig, L. et al. 1997. Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. Proc. Natl. Acad. Sci. USA 94:8393-8398.

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

ENGINEERING PLANTS TO MANAGE STRESS
In their quest to feed the ever-increasing world population, agricultural scientists have to contend with the dry reality that arable land on this earth is very limited. Much of the terra firma is inhospitable to farming because of high salt, dry or frigid conditions. Even large tracts of land currently under agricultural cultivation around the world suffer from these maladies that limit crop productivity. If crops can be redesigned to better cope with stress, agricultural production can be increased dramatically. Although impressive strides have been made in engineering crops resistant to diseases and pests, making them hardier to drought and salt conditions has been more challenging. Many plants such as the cactus which brave the arid deserts of Arizona or Atacama do so because of a multitude of complex adaptive mechanisms which are yet intractable to gene manipulation. Nevertheless, molecular biologists are zeroing in on important secrets of organisms that tolerate salt or drought conditions, and using this knowledge to develop hardier crops.

One example is a recent report from Japan, where tolerance to salt and cold stress was engineered in plants by enabling them to accumulate glycinebetaine (1). Betaine, as it is also called, is found in organisms as diverse as bacteria, spinach and humans. Betaine is thought to insulate plant cells against the ravages of salt by preserving the osmotic balance, by stabilizing the structure of proteins such as rubisco and by protecting the photosynthetic apparatus. The enzyme choline oxidase helps in the production of betaine from choline.

A group led by Norio Murata at the National Institute of Basic Biology in Okazaki has cloned a gene for choline oxidase (codA) from a soil bacterium. They had observed earlier that cyanobacterium engineered with this gene tolerated saline and cold conditions. They then developed transgenic Arabidopsis plants with the codA gene fused to a transit peptide that directed the enzyme into chloroplasts. When seeds from transformed plants were tested under high salt conditions (200 to 300mM NaCl), most germinated well while regular seeds did not.

Increased salt tolerance due to betaine accumulation was also observed in germinated seedlings and adult plants. Nonengineered adult plants died quickly when transferred to salt conditions (200mM NaCl), but transgenic plants with the codA gene continued to grow, albeit slowly. Engineered plants producing choline oxidase also showed increased resilience to damaging cold exposures. With increasing salt and cold conditions, transgenic plants maintained photosynthetic activity while control plants ceased such activity under stress. The Murata group has now extended this research to real world crops and has developed rice plants with the stress-tolerant gene. Studies with rice confirmed that chloroplast-targeting of the codA gene was a smart move as non-targeted transgenic plants were less tolerant to stress.

In addition to betaine, other compatible solutes such as mannitol and proline also promote drought tolerance in plants, and plants engineered to produce these compounds were also stress tolerant. The Japanese study further extends the horizons of the plant stress research, and collectively these studies foretell a scenario where biotechnology would arm our future crops with new tactics to survive in hostile environments.

Reference

1. Hayashi, H. et al. 1997. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. The Plant Journal 12:133-142.

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

ANIMAL RESEARCH NEWS

HOW NOW GREEN COW?
The low efficiency of transgenic animal production by microinjection remains a major problem for the development of genetically engineered livestock. Typically only 10% of the animals born after embryo microinjection are transgenic. Thus large numbers of both donor and recipient animals need to be maintained at considerable expense to ensure the production of a transgenic animal. If a method could be developed to prescreen microinjected embryos for transgene expression prior to transfer into a surrogate mother, then the frequency of producing transgenic animals would increase. This would reduce the number of animals needed to serve as embryo recipients and would reduce the cost of maintaining pregnancies that will not produce transgenic progeny.

Researchers at the National Children's Medical Research Center in Japan have reported that prescreening mouse embryos that expressed a microinjected green fluorescent protein (GFP) construct greatly increased the frequency of transgenic mice born (1). Green fluorescent protein emits bright green light upon exposure to ultraviolet light in the absence of any substrates or cofactors and serves as a highly sensitive marker of gene expression.

From 55 GFP-positive blastocysts, eight fetuses and four live-born mice were obtained. Eleven out of the 12 mice were transgenic by PCR and eight of these were also positive by Southern blot. The GFP-transgenic mice developed normally demonstrating that GFP expression was not deleterious. The Japanese researchers also reported that GFP was expressed in microinjected bovine embryos, although these embryos were not transferred to recipients.

Preimplantation genetic diagnosis by PCR has been successfully performed on a single cell biopsied from an 8-cell mammalian embryo with minimal effect on the viability or rate of embryo development. This method, however, is not applicable for screening microinjected oocytes for the presence of an integrated transgene. PCR cannot distinguish between transgenes that are integrated into the genome and those that remain unintegrated. This is a problem because unintegrated DNA has been shown to persist in a developing embryo for numerous cell divisions before it is lost. In addition, integration of the transgene may not occur at the one-cell stage but at the multi-cell stage. As a result the embryo is a mosaic, with some of the cells containing the integrated transgene and other cells lacking it. Subsequent analysis of one biopsied cell may result in false positives or false negatives. Thus it is somewhat surprising that the use of GFP as a marker gene for prescreening transgenic animals worked so well. If this method proves equally efficacious for livestock, then it would provide a valuable tool for greatly improving the efficiency of producing transgenic livestock and would result in a significant savings in development costs.

Reference

Takada, T., Iida, K., Awaji, T., Itoh, K., Takahashi, R., Shibui, A., Yoshida, K., Sugano, S. and Tsujimoto, G. (1997). Selective production of transgenic mice using green fluorescent protein as a marker. Nature Biotechnology 15: 458-461.

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

VACCINATING ANIMALS TO CONTROL INSECT PESTS
Disguised by a lovely name, Lucilia cuprina is an insidious pest. Larvae of this blowfly can infest the skin of sheep and goats, feeding on dermal tissue, tissue fluids, and blood. Severe infestations result in a flock of miserable animals and considerable economic losses to the sheep industry. Current methods of chemical pest control are becoming inadequate as insects develop resistance to pesticides and concerns grow about chemical residues contaminating livestock products and the environment. Alternative methods of controlling insect pests are needed.

Researchers from the Commonwealth Scientific and Industrial Research Organization (CSIRO) in Australia recently report a novel method of immunological pest control. The method involves vaccinating the animal host with a protein extracted from the gut membrane of the insect pest. Antibodies to the insect protein are raised by the animal's immune system and circulate in its blood. Fly larvae infesting the animal ingest the antibodies as they feed.

In laboratory bioassays, L. cuprina larvae that were allowed to feed on the blood of an immunized animal showed a 60% reduction in growth rate. Growth inhibition is thought to result from a restricted uptake of nutrients in the larvae, caused by binding of the ingested antibodies to gut membranes and the subsequent formation of an impervious layer of undefined composition.

When growth rate was reduced by 60%, the starved larvae showed no observable external signs of damage and no reduction in survival. A reduction in larval survival was only observed after concentrating the anti-gut protein immunoglobulin to a level that caused greater than 80% growth inhibition. The resulting undersized larvae are slow to develop into pupae but continue to develop normally once they reach that stage. It is the delay in reaching pupation that reduces survival because the larvae are more susceptible to natural mortality during this time.

In laboratory tests this method of immunocontrol appears promising, however, the ultimate test will be whether or not it works in a barnyard or field setting. For this approach to be effective, it is necessary to induce a strong immune response in order to significantly retard the growth rate and thereby reduce the survival of larvae feeding on immunized sheep. Successful cloning of the gene encoding the gut protein by the same research group opens up the possibility of inducing an immune response by injecting DNA directly into tissues.

This immunocontrol strategy differs from the more conventional uses of vaccination in which induced antibodies bind to the surface of a pathogen and mark it for removal by the immune system. Instead, the antibodies are like the soldiers hidden in the Trojan horse --- the feeding pest unwittingly takes them inside whereupon they attack from within. The approach has potentially wide applicability to controlling other insect pests that feed directly on mammalian blood or tissues.

An even more interesting prospect is applying the strategy to protect plants. Transgenic plants have been shown to be capable of producing large quantities of recombinant antibodies. Perhaps crops could be engineered to express antibodies directed against gut proteins of their major insect pests. A potentially significant secondary benefit would be that, by providing a completely new mechanism of insect control, use of immunocontrol would help preserve the utility of other strategies such as the use of Bt genes. Possibilities like this are an encouraging development in the never-ending battle to control pests.

Reference

Casu, R., Eisemann, C., Pearson, R., Riding, G., East, I., Donaldson, A., Cadogan, L. and Tellam,R. (1997). Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host. Proc. Natl. Acad. Sci. USA 94:8939-8944.

Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
traynor@nbiap.biochem.vt.edu

INDUSTRY NEWS

NOVARTIS AND CHIRON FORM ALLIANCE FOR COMBINATORIAL CHEMISTRY
Combinatorial chemistry is most commonly known as the process of synthesizing, screening, and optimizing a large number of compounds for drug discovery (1). But this technique is not the unique domain of biopharmaceutical pioneers, as explorers in the field of agriculture are beginning to harness its power for new product discovery. Novartis Crop Protection AG, a sector of the life sciences multi-national Novartis, recently announced a three-year agreement with Chiron Technologies involving the use of combinatorial chemistry to generate compounds with potential crop protection and animal health applications (2).

Combinatorial chemistry uses highly automated procedures to synthesize large collections (or libraries) of new compounds rapidly by combining numerous different molecular building blocks of existing or novel chemical agents (2). One of the key factors driving the interest in combinatorial chemistry is the increased speed in which new compounds can be produced and optimized for use in ever evolving high-throughput screening procedures used to identify desired activity. Increased production and optimization speed means less time and expense for companies faced with constantly escalating research and development costs. Where traditional chemical synthesis techniques allowed a company to synthesize a few hundred compounds a year, combinatorial chemistry allows a firm to create thousands of new compounds in a week.

An example taken from the drug development realm demonstrates the power of this technique. Traditional medicinal chemists at Pfizer have synthesized approximately 300,000 compounds over the last 40-50 years, in contrast to their creation of more than 500,000 compounds within a 4-6 week period using combinatorial chemistry (3). Trega Biosciences (formerly known as Houghten Pharmaceuticals), another company using combinatorial chemistry in its drug development program, uses a technique which allows the synthesis of thousands of related molecules simultaneously at a cost of approximately 50 cents per compound (2).

Under the new agreement, Novartis will support Chiron's chemical synthesis group in Australia. The group will produce selected combinatorial libraries which Novartis will then screen. Novartis will have an option to an exclusive, worldwide license to active compounds and derivatives for crop protection and animal health uses. Chiron will receive up-front fees, as well as annual and milestone payments. In addition, Chiron will be paid royalties on any products arising from the arrangement (2). Novartis will be able to accelerate its internal lead finding activities through this new agreement, which is being touted as one of the first research collaborations to bring the new skills of combinatorial chemistry into agribusiness.

Alliances such as this one, and the recent deal between CuraGen and Pioneer Hi-Bred (described in the July ISB News Report) demonstrate the significant investment that agricultural companies are making in the newer "platform technologies" such as combinatorial chemistry and genomics, to accelerate their new product discovery efforts.

References

1. Chovav, Meirav. Houghten Pharmaceuticals. "Tea"-ing off with combinatorial chemistry. Salomon Brothers research report, June 12, 1996.

2. Press release (http://www.novartis.com). Novartis and Chiron sign agreement on combinatorial chemistry for crop protection research. September 9, 1997.

3. Glaser, V. Companies Develop Novel Technologies for Maximizing Combinatorial Library Potential. Genetic Engineering News, Vol. 16, No. 10, May 15, 1996, pp.1, 19.

William O. Bullock
Institute for Biotechnology Information
http://www.biotechinfo.com


The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. It does not necessarily reflect the views of the U.S. Department of Agriculture or of Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge. P.L. Traynor, Editor.

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