IN THIS ISSUE:
What’s In the Commercial Pipeline?
Coming Attractions At ISB
Enzymatic Detoxification Of Mycotoxins In Transgenic Maize
Engineered Plants Show Promise As Edible Vaccine For Diabetes
Producing Reagent Proteins In Plants
Largest Segment Of Plant DNA Sequenced To Date
Alcohol-Induced Expression In Transgenic Plants
Big Blue Advancing In Genomics Arena

NEWS AND NOTES

WHAT'S IN THE COMMERCIAL PIPELINE?

Four new transgenic crops are making their way through USDA's regulatory review process. The Animal and Plant Health Inspection Service (APHIS) recently announced that a genetically modified line of canola has been deregulated, and three more Petitions for Determination of Regulated Status have been received, two for corn and one for sugar beet. Notices are published to inform the public that APHIS will accept written comments regarding a Petition from any interested person for a period of 60 days from the date of the notice.

APHIS regulations concerning "Introduction of Organisms and Products Altered or Produced Through Genetic Engineering Which Are Plant Pests or Which There Is Reason to Believe Are Plant Pests," regulate the introduction (importation, interstate movement, or release into the environment) of genetically modified organisms that are known or potential plant pests. APHIS' definition of a plant pest is very broad, and covers direct or indirect injury, disease, or damage not just to agricultural crops, but also to plants in general, for example, native species, as well as to organisms that may be beneficial to plants (i.e. honeybees, rhizobia, etc.). Such organisms and products are normally considered "regulated articles," and are subject to restrictions on their transport and release under Federal law, but any person may submit a petition to APHIS seeking a determination that an article should not be regulated.

After the comment period closes, APHIS reviews the data submitted by the petitioner, all written comments received during the comment period, and any other relevant information. Based on the available information, APHIS furnishes a response to the petitioner and publishes a notice in the Federal Register announcing the regulatory status of the subject plants.

Glufosinate-tolerant canola. On September 30, 1997, APHIS published a notice in the Federal Register announcing that AgrEvo USA Company had petitioned for a determination of nonregulated status for canola (Brassica napus L.) which has been genetically engineered for tolerance to the herbicide glufosinate. The canola contains a pat gene derived from Streptomyces viridochromogenes, under the control of the 35S promoter and terminator derived from the plant pathogen CaMV.

APHIS received no comments on the petition during the designated 60-day comment period. Based on its analysis of the data submitted by AgrEvo, and a review of other scientific data and field test reports of the subject canola, APHIS has determined that it: (1) exhibits no plant pathogenic properties; (2) is no more likely to become a weed than canola developed by traditional breeding techniques; (3) is unlikely to increase the weediness potential for any other cultivated or wild species with which it can interbreed; (4) will not cause damage to raw or processed agricultural commodities; and (5) will not harm threatened or endangered species or other organisms, such as bees, that are beneficial to agriculture. The USDA deregulation became effective January 29, 1998.

Insect-resistant and glufosinate-tolerant corn. A petition for determination of nonregulated status received on September 22, 1997 from AgrEvo USA Company. The corn described in the petition has been genetically engineered to express a Cry9C insecticidal protein derived from the common soil bacterium, Bacillus thuringiensis subsp. tolworthi. The Cry9C protein is effective in controlling larvae of the European corn borer during the complete growing season. The corn also contains the bar gene derived from the bacterium Streptomyces hygroscopicus. The bar gene encodes phosphinothricin acetyl-transferase, an enzyme which confers gluphosinate tolerance. Although the corn contains the bla selectable marker gene which is normally expressed in bacteria, tests indicate that this gene is not expressed in the plant.

Written comments on this petition must be received on or before April 24, 1998. Refer to Docket No. 97-119-1.

Male sterile glufosinate-tolerant corn. A petition for determination of nonregulated status was received on December 8, 1997 from Pioneer Hi-Bred International, Inc. The petition describes three corn lines that have been genetically engineered to contain a DNA adenine methylase enzyme (or dam gene) derived from E. coli. The dam gene expresses in a specific plant tissue, which results in the inability of the transformed plants to produce anthers or pollen. The corn lines also contain the phosphinothricin acetyltransferase (pat) selectable marker gene isolated from the bacterium S. viridochromogenes, which, when introduced into a plant cell, confers tolerance to the herbicide glufosinate. Linkage of the dam gene, which induces male sterility, with the pat marker gene enables identification of the male sterile line for use in the production of hybrid seed.

Written comments on this petition must be received on or before April 20, 1998. Refer to Docket No. 98-009-1.

Glufosinate-tolerant sugar beet. A petition for determination of nonregulated status was received on December 2, 1997 from AgrEvo USA Company. The petition describes a sugar beet line that has been genetically engineered to contain a synthetic version of the pat gene derived from Streptomyces viridochromogenes. Expression of the pat gene is controlled by the 35S promoter and terminator sequences derived from CaMV. The sugar beets also contain an aph(3')II or nptII marker gene used in plant transformation. Expression is controlled by gene sequences derived from Agrobacterium tumefaciens, and analysis indicates that the NPTII protein is expressed in certain parts of the plants.

Written comments on this petition must be received on or before April 7, 1998. Refer to Docket No. 97-130-1.

Sending comments. Comments (an original and three copies) may be sent to: Docket No. (see individual numbers above), Regulatory Analysis and Development, PPD, APHIS, Suite 3C03, 4700 River Road Unit 118, Riverdale, MD 20737-1238. Please state the Docket No. in your comments. To obtain a copy of a petition or notice, contact Ms. Kay Peterson at 301-734-4885, mkpeterson@aphis.usda.gov.

Jim Westwood
Virginia Tech
westwood@vt.edu

New Look for the ISB Website
The newly redesigned ISB website (http://www.isb.vt.edu) will be unveiled in March. The new look incorporates side-by-side menu and document windows that make it easier to access our growing collection of online information resources.

The Environmental Releases database now contains over 400 field test environmental assessments. There are also more than 1200 News Report articles which can be searched by keywords to track research developments over the past ten years.

Web Version of Fish Performance Standards
Also in March we plan to bring online a web-based version of the "Performance Standards for Safely Conducting Research with Genetically Modified Fish and Shellfish". This version, which includes graphics, will be useable by anyone with a standard web browser. The program's structure is readily adaptable to risk assessment / risk management evaluations of other transgenic organisms. ISB welcomes inquiries regarding joint efforts to create such decision support tools.

Doug King
Information Sytems for Biotechnology
nbiap@vt.edu

PLANT RESEARCH NEWS

ENZYMATIC DETOXIFICATION OF MYCOTOXINS IN TRANSGENIC MAIZE

Moldy grain looks bad, smells bad and tastes bad. But when the poor quality is due to certain types of molds and fungi, the presence of toxic compounds produced by the fungus can make the situation far worse. Fumonisins, for example, are a family of highly stable, toxic molecules produced by Fusarium moniliforme and related mold species found in most maize-growing regions of the world. Fumonisin is toxic to livestock, particularly horses, carcinogenic in rats, and occurs widely in maize grain and some maize products. High levels may be found in visibly moldy grain or "screenings", and even clean looking grain can accumulate the toxic compound.

It's been estimated that 25% of the world's food crops are affected by mycotoxins. Although actual losses are difficult to quantitate, grain producers see reduced yield and marketability, animal producers see reduced performance, grain handlers and distributors bear costs of monitoring and loss of markets, processors see product loss, consumers pay higher costs and endure chronic health effects, and society bears the cost of regulatory procedures and tariff and trade effects.

Jon Duvick, in the Crop Protection Department at Pioneer Hi-Bred, International, reported on their efforts to address the problem of fumonisin in corn using a strategy applicable to a variety of mycotoxins in various crops. The approach was to identify functional groups involved in biological activity of the toxin, isolate fumonisin-metabolizing microbes from moldy ears and stalks, identify fumonisin metabolizing enzymes, clone the genes and transform them into corn.

From washings of moldy kernels, the group identified 17 isolates of filamentous black yeast fungi and one yellow-pigmented Gram negative bacterium, species unknown. A radio-labeled fumonisin (FB1) was used to unravel the catabolic pathways. Genes encoding key enzymes were cloned, and their activity was confirmed in heterologous expression systems.

Transgenic lines showed promising results in greenhouse ear mold assays and 1997 field tests. While these preliminary results support the validity of the approach, Duvick noted that technical challenges remain, and significant regulatory issues will need to be addressed before mycotoxin-free corn is a reality.

Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu

ENGINEERED PLANTS SHOW PROMISE AS EDIBLE VACCINE FOR DIABETES

We all know some one who has diabetes. There are 16 million people with diabetes in the U.S. alone and more than 100 million worldwide. A study from Canada now provides a ray of hope that plants can be bioengineered to fight this silent killer. Diabetes is among the leading causes of death and shortens life expectancy by 20 years. Health care expenditures in the U.S. for diabetics exceed $130 billion, and a child diagnosed with this disease can expect to spend $600,000 over her life time.

Type I diabetes, also known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, primarily affects children or young adults and accounts for 5-10% of the diagnosed diabetes in North America. It is an autoimmune disease where the pancreatic beta cells that produce insulin are destroyed by the body's own immune system. As a result, glucose builds up in the blood, overflows into urine and thus the body loses its main source of fuel. People with IDDM inject themselves with insulin up to six times a day and may check their blood glucose level six to eight times a day.

Research by Shengwu Ma, Anthony Jevnikar and colleagues at the John P. Robarts Institute in the University of the Western Ontario (Canada) shows that diabetes can be prevented in mice by feeding them with plants engineered to produce a diabetes-related protein (1). "The idea is based on 'oral tolerance' where the autoimmune system is selectively turned off early by teaching the body to tolerate the antigenic proteins", says Jevnikar. The pancreatic protein glutamic acid decarboxylase (GAD67) is linked to the onset of IDDM, and when injected into mice it is known to prevent diabetes.

However, clinical studies on oral tolerance require large quantities of protein, which is impractical to produce in bacteria or mammalian cells and if extracted from animals would be prohibitively expensive. The Canadian scientists thus turned to plants because "they are ideal as they can synthesize, glycosylate and assemble proteins to provide huge quantities of soluble proteins at relatively low cost", says Ma, a plant molecular biologist. Plants can be eaten directly and without cooking or heat sterilization, as heat destroys the protein, and they do not carry risks of pathogens from animal sources.

The Canadian group developed transgenic potato and tobacco plants with a gene for GAD67, and fed them to non-obese diabetic mice, which develop insulin-dependent diabetes spontaneously. The results were intriguing: only 20% of the pre-diabetic mice fed with transgenic plants developed diabetes while 70% of the non-treated mice developed the disease. The treated mice also showed increased levels of IgG1, an antibody associated with cytokines which suppress harmful immune responses. Thus, the antigen produced in plants appears to retain immunogenicity, dampen the destructive process of the immune system and prevent diabetes in an animal model. According to Ma, this is the first proof of principle for the use of an edible vaccine in the treatment of an autoimmune disease.

Although the response from the scientific community has been enthusiastic, the researchers are being prudently cautious about their findings. There are many hurdles to overcome before human clinical trials can begin. More animal studies are needed, expensive surveys must be undertaken to identify potential human candidates, possible side effects need to be determined and then there is the problem of dosage control. Further, there is no proof yet of a therapeutic role of oral tolerance in human disease and there is also looming concern that orally ingested autoantigens might worsen some diseases (2). To target children it may be necessary to produce edible vaccines in tomato or banana.

Jevnikar and Ma clearly recognize these challenges ahead but believe the success of their work will have wider implications. They note that "molecular pharming" to produce autoantigens in plants may also target other autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, lupus and even transplant rejection, and have intiated work in some of these directions.

References

1. Ma, S. W. et al., 1997. Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nature Medicine 7:793-796

2. Blanas, E. et al.,1996. Induction of autoimmune diabetes by oral administration of autoantigen. Science 274:1707-1709.

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

PRODUCING REAGENT PROTEINS IN PLANTS

There's an interesting new product in the 1998 Sigma catalog of Biochemicals and Reagents for Life Science Research (page 158). Avidin, a chicken egg white protein used as a key reagent in numerous diagnostic kits and procedures, is now available as a recombinant protein expressed in corn. The new catalog item joins bacterial beta-glucuronidase, or GUS (page 530), another commonly used reagent protein for which a transgenic plant product became commercially available last year. Both reagent proteins were developed by ProdiGene, Inc. (College Station, TX), a company that uses genetic engineering to create non-commodity crops.

Corn was engineered with an avidin gene under the control of a ubiquitin promoter complete with intron, fused with a peptide targeting sequence that directs the newly synthesized protein to the cell wall. In 1994 and 1995 field grown plants, avidin accumulated in transgenic seed as 2-3% of extractable protein, ranging between 200 and 300 mg per kilogram seed, or around 450 grams per acre. Slightly more than half the avidin in kernels is found in the embryo, about a fifth is in the endosperm, and the rest is lost during dry milling.

The reagent is produced by affinity purification of soluble protein extracted from flaked seed meal. Its commercial value is evident in a comparison of raw material needed to obtain 20 grams: a ton of eggs ($1000) vs. four bushels of corn ($20). Relative costs of storage, transportation and processing are also favorable.

Because of the high yield and value, only limited acreage is needed to supply Sigma with the recombinant product. ProdiGene grows the engineered corn under a USDA/ APHIS field test permit, and is unlikely to seek deregulation any time soon. The corn is grown as an identity-preserved crop.

An unexpected offshoot of the research is the discovery that very nearly all the avidin expressing plants are male sterile. Although the mechanism has not been examined yet, presumably the expressed avidin binds to biotin in plant cells; it may be that pollen formation is more sensitive to this than other biological functions in the plant.

The effect has at least three implications according to John Howard, President and CEO of ProdiGene. First, it could be useful as a source of male sterility in the production of conventional hybrid seed corn, an application likely to be of interest to major seed producers. Secondly, if the avidin market ever justifies large scale production of ProdiGene's transgenic lines, they would have to modify their plantings to include fertile males in order to get seed set. The third implication is that avidin-induced male sterility potentially could be a useful risk management tool where the release of pollen from transgenic corn raises environmental or regulatory concerns.

Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu

LARGEST SEGMENT OF PLANT DNA SEQUENCED TO DATE

Ten years ago few people had heard of Arabidopsis but now the weed is a favorite of plant molecular biologists. With its compact genome - at 120 million base pairs (Mb) it's only a tenth that of many crop plants - petite size, and hurried life cycle, Arabidopsis lends itself neatly to genome studies. It is easier to identify and isolate genes from this model plant than genetically more complex crop plants.

A significant piece of the Arabidopsis genome has now been sequenced and analyzed, as reported by Michael Bevan and coworkers in the journal Nature (1). The 1.9 Mb catalog (on the Internet at http://www.mips.biochem.mpg.de/mips/athaliana/) provides 'a tantalizing preview of the bricks and mortar needed to build a plant' says Joseph Ecker of the University of Pennsylvania (2).

The mammoth effort of sequencing the largest contiguous plant DNA so far involved 68 scientists from 21 institutions located in nine countries spanning the Atlantic. Although this sequence is just a small fraction of the total, it provides an intriguing glimpse into the architecture of a higher plant genome. Among the 389 genes that are packed densely in the region, over half are already known to be present in other organisms, while the function of 46% of the genes is unknown. The study confirms an earlier prediction that Arabidopsis has 21,000 protein-coding (active) genes.

The entire genome is expected to be sequenced through collective international effort by the year 2004. This may be accomplished even sooner because of a new funding initiative by the U.S. Congress.

References

1. Bevan, M. et al., 1998. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391:485-488.

2. Ecker, J. E. 1998. Genes blossom from a weed. Nature 391:438-9.

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

ALCOHOL-INDUCED EXPRESSION IN TRANSGENIC PLANTS

Promoters are master switches that determine the location, timing and intensity of gene expression. The popular '35S' promoter from cauliflower mosaic virus (CaMV) is a plant promoter that has been widely used in the development of transgenic plants. While the CaMV35S is powerful, it is constitutively expressed, i.e., it is turned on all the time and throughout the plant. In cases where gene expression needs to be tailored to a select organ, say for instance the tuber in potato, tissue-specific promoters such as the one from the patatin gene are employed.

There has always been a need for plant promoters which could be activated at will. Some transgenes are known to impact plant growth or yield, so it is prudent to turn them on only when needed. Examples include inducing male sterility for hybrid seed production without the need for a restorer line, turning on disease resistance genes only when the pathogen appears to counter pathogen adaptation, or delaying expression of transgenes when the new protein interferes with early plant growth.

There are few, if any, options for conditionally expressing genes under field conditions. Promoters responsive to chemicals such as tetracycline and dexamethasone are available, but they are impractical for use in the field. Now, a group from Europe has come up with a solution where the scientist - or the farmer in the field - can turn on gene expression when needed (1). This control will rely on the use of a simple and inexpensive compound to induce gene expression: alcohol, a biodegradable and natural organic molecule with minimal toxicity on plants.

The group, led by Brian Tomsett and Mark Caddick of the University of Liverpool (UK), employed the regulatory sequences of the alcA gene (encoding alcohol dehydrogenase I) from the filamentous fungus Aspergillus nidulans. This gene is turned on by ethanol and its promoter is widely used to overexpress proteins in fungi. To be certain of strong expression in plants, Tomsett and coworkers added a few critical elements of the CaMV promoter and attached it to the chloramphenicol acetyl transferase (CAT) marker gene, and introduced the cassette into plants. Untreated transgenic plants showed no CAT expression but turned on the gene when 0.1% ethanol was applied to roots, thus showing that the alc regulatory system works in plants.

The researchers then focused on an application of practical importance. Invertase is an enzyme known to impact carbon flux in plants and thus is of broad interest in fundamental and applied research. When the invertase gene is expressed under the control of CaMV promoter, the plants are extremely stunted and turn chlorotic, as invertase can interfere with the metabolism of sink leaves. Transgenic plants expressing the invertase gene under the control of alcA promoter, however, developed normally. Only when the roots were drenched with alcohol was the invertase expressed, leading to clear signs of chlorosis. The gene product was seen as early as six hours after alcohol treatment.

Caddick and coworkers justifiably conclude that the alc system will prove an invaluable tool in the study of essential genes and pathways in higher plants. It does not have much 'leaky' expression and is not turned on by the plant's own alcohol, produced when waterlogged. Christiane Gatz of the University of Gottingen, an expert on chemically inducible promoters in plants, says that the alc system may "develop into one of the most broadly applicable regulatory systems for plant gene expression currently available" (2). Gatz is also optimistic that inducers less volatile than ethanol may work with the alc system, which she says has a "tremendous potential to take plant gene expression to an intoxicating new high".

References

1. Caddick, M. X., et al. 1998. An ethanol inducible gene switch for plants used to manipulate carbon metabolism. Nature Biotechnology 16:177-180.

2. Gatz, C. 1998. An intoxicating switch for plant transgene expression. Nature Biotechnology 16: 140.

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

INDUSTRY NEWS

BIG BLUE ADVANCING IN GENOMICS ARENA

IBM has long been known for information technology products and services, and more recently has been applying its IT capabilities to the area of computational biology. This field combines mathematics with biological sciences to provide new avenues for the design and synthesis of novel products.

One key area of IBM's research with bioscience applications is the development of algorithms, which are basically sets of instructions for carrying out particular tasks. In computers, algorithms are usually encoded in a computer language instruction set that manipulates data. The use of algorithms in pattern recognition is applicable to genome analysis, in which the technology is used to sort through massive amounts of data to identify repeated patterns. This technology has been applied in scanning the billions of bits of human genome data already accumulated.

One specific technology that IBM is working on is FLASH (Fast Look-up Algorithm for Structural Homology). FLASH focuses on recognizing items that are similar but not identical to objects in a database. It looks for matches only in places where it is likely to find them and ignores the rest of the database. FLASH is being used to match new or unidentified DNA and protein sequences with existing sequences whose functions are known, thus serving as a powerful tool in elucidating function from structure.

This type of algorithm technology is the basis of a recently announced strategic alliance between IBM and Monsanto. Scientists from the two companies will work to develop information technologies to identify and map the genetic structure of major plant groups and human disease organisms. The actual algorithm technology that will be used for this research is known as Teiresias, a next-generation pattern discovery algorithm developed by IBM Research. The technology is expected to significantly speed discovery of hidden patterns in DNA and protein sequence databases, with the intent of accelerating and expanding Monsanto's life science product development.

Given the interest being paid to genomics by agricultural and pharmaceutical companies as a key enabling technology for future product discovery and development, it will be worth watching to see if IBM enters into future collaborations with the industrial life science sector in this area.

References

1. IBM genomics collaboration, Nature Biotechnology, Vol 16, Feb. 1998, p. 122.

2. IBM Internet web site (http://www.ibm.com).

William O. Bullock
Institute for Biotechnology Information, LLC
Research Triangle Park, NC
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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