TRANSGENIC ARTHROPOD SYMPOSIUM BEING PLANNED
The next national meeting of the Entomological Society of America will have a symposium concerned with the release into the environment of genetically engineered (transgenic) arthropods. The program, which is being organized by the USDA-APHIS Transgenic Arthropod Team and the Information Systems for Biotechnology project of the National Biological Impact Assessment Program, is in the early stages of development for the December, 1996 meeting.
The organizers are very interested both in suggestions for topics and speakers, and in learning of the availability and interest of individuals to participate in the session. We presently envision a 4 hour morning session, directed toward non-molecular biologists, that would cover such topics as the recent history of transgenic organism releases, molecular biology and methodology associated with the production of transgenic arthropods, environmental issues associated with their release, descriptions of current projects, a regulatory overview, and future directions.
Individuals wishing to contribute to the planning effort, or to nominate themselves as presenters, please contact either Orrey Young (301-734-7612; oyoung@aphis.usda.gov) or Pat Traynor (540-231-2620; traynor@nbiap.biochem.vt.edu) before March 15, 1996.
A cotton line genetically engineered for tolerance to sulfonylurea herbicides is deemed safe to grow without further USDA permits or acknowledged notifications. The cotton line, designated 19-51a, is produced by Dupont Agricultural Products in Wilmington, Delaware.
The transgenic cotton was regulated because it contains gene sequences derived from the plant pathogen A. tumefaciens. The line has been evaluated in field trials conducted under APHIS permits or notifications since 1991. After reviewing the biology, propagation and cultivation of the plant, the source of the engineered genes, the vector used to transfer them and the stability of the insertion, APHIS concluded that cotton line 19-51a and any progeny derived from hybrid crosses with other transformed cotton varieties will be just as safe to grow as traditionally bred cotton lines that are not regulated.
The determination and environmental assessment documents for this petition, and for all other petitions granted by APHIS, are available on the internet at http://www.aphis.usda.gov/BBEP/BP/. Printed copies may be obtained from Kay Peterson USDA/APHIS/BBEP, 4700 River Road, Unit 147, Riverdale, Md. 20737-1237 (301-734-7612).
Pat Traynor
Information Systems for Biotechnology
Newsletters of the United Kingdom's Advisory Committee on Releases to the Environment (ACRE) are online at http://www.shef.ac.uk/~doe. ACRE gives advice to the Secretary of State about human and environmental safety in the context of releases of genetically modified organisms (GMOs) into the environment.
Newsletter No. 4 contains a complete survey of the 78 applications for field testing in the U.K. filed since February, 1993. A quick reading shows the following distribution of crops and traits:
The list continues down through wheat, tobacco, corn, chicory, eucalyptus, ending with one application each for strawberry, apple, and tomato. Tables show the crop, introduced trait, size of the release area, isolation distances used or other means of conferring isolation, and time and frequency of monitoring and reporting.
ACRE has published a series of Guidance Notes on regulations, biosafety, and risk assessment for the release of GMOs that are available for a nominal charge. Ordering information and contacts can be found on the homepage.
Pat Traynor
The European Commission is spending 1 million ECUs ($US 1.24 million) over the next three years on advancing public awareness and understanding of biotechnology across Europe. About half the money is going to the European Federation of Biotechnology Task Group on Public Perceptions of Biotechnology. A further third of the funding will be used to coordinate international opinion research in association with the Task Group.
The European Federation of Biotechnology is the central, Europe-wide organization for biotechnology. It incorporates more than 80 biotechnology-related scientific societies. Established in 1991, its Task Group on Public Perceptions of Biotechnology has 50 leading members from Europe's biotechnology industry and research, communications and survey research, the media, and environmental, consumer and patients' organizations.
The overall aim of the Task Group is to promote greater public awareness and understanding of biotechnology and to encourage informed, open debate. This is considered essential if European biotechnology is to remain competitive worldwide, while at the same time retaining public confidence and trust. The Task Group uses several approaches:
This money will enable the expansion of these activities and also new targeted activities for specific groups: industrialists, environmentalists, consumers etc. Key emphasis will be on the Task Group's position as the independent intermediary for all the varied parties concerned with - and by - biotechnology.
Further information is available from Professor John Durant (Task Group chairman), Assistant Director, The Science Museum, Exhibition Road, London SW7 2DD, Great Britain. Tel: +44 171 9388201; fax: +44 171 9388213
Editor's Note: The following two articles are based on material from the March 1996 issue of Agricultural Research magazine, published by USDA-ARS and supplied by Jim DeQuattro, Current Information, ARS; 301-344-2311; jdequatt@asrr.arsusda.gov
A new mathematical tool developed for farm biotechnology may also help medical researchers devise gene therapies for people. USDA Agriculture Research Service (ARS) scientists and a software engineer at Silicon Graphics, in Mountain View, California, devised a new algorithm to scan computer data of genetic code. The algorithm seeks patterns occurring in genetic material associated with jumping genes, or transposons.
To give the algorithm a tough challenge, the scientists tested it on a human gene database -- far larger than the plant gene database. The algorithm found two sequences that are among the first DNA transposons detected in the human genome (Nature, Dec. 14, 1995, p. 672). Most known human transposons are the RNA, or single-stranded, type. Transposons found in plant genomes may provide elements that can be used to insert new genes into agronomically important crop species.
ARS scientists are hoping that a common, soil-dwelling fungus can find a new role as a versatile, lower cost workhorse for biotechnology. They used the Olpidium zoospore fungus to shuttle markers genes into wheat, and have patented the process for transporting new genes into plants. The work, first published in Phytopathology in 1994 (vol.84:684-687) is still generating interest as a gene delivery system.
Currently, the most commonly used transformation methods rely on either Agrobacterium or a "gene gun" to introduce foreign DNA into plants. The choice often depends on the species of plant, the tissue to be transformed, and the availability of suitable vectors. Use of Olpidium adds another option to the list.
As a genetic courier, the fungus delivers DNA into plants within an envelope -- the harmless outer coat of tobacco necrosis virus. The virus normally spreads from plant to plant via the fungus which is associated with the root tips. ARS researchers purified the virus coat protein subunits, then mixed the protein with plasmid DNA under conditions that allowed stable pseudo virus particles to reassemble. In the soil, the particles were picked up by germinating fungal zoospores and passed into root tips. Once inside plant cells, the pseudo virus particles came apart allowing a marker gene on the enclosed plasmid to be transiently expressed.
Although the ARS group is not able to pursue this line of research, they have received a number of inquiries from others who may. Olpidium could just as easily deliver pseudo virus particles containing DNA sequences that direct stable integration into the plant genome, and thus be a new mechanism for plant transformation. The wide host range of the fungus, encompassing species ranging from grasses to broadleaf plants, may make it a useful vector for beans, lettuce, and many other crops. For more information, contact Lingyu Zhang and William Langenberg, USDA-ARS Wheat, Sorghum, and Forage Research Laboratory, Lincoln, Nebraska; 402-472-3162.
PLANT BIOTECHNOLOGY RESEARCH AT TUSKEGEE
UNIVERSITY
The Center for Plant Biotechnology Research at Tuskegee University aims to improve the productivity of crops such as sweet potato, peanut, cowpea and muskmelon using molecular and cellular genetic approaches. We also provide training in genetic engineering to under-represented U.S. minority students and scientists from developing countries.
The Center continues along the tradition of George Washington Carver, the legendary scientist who developed hundreds of innovative products from sweet potato and peanut while working at Tuskegee University earlier this century. Sweet potato is nutritionally and economically very important to many people around the world, especially those with limited resources. It produces more calories and protein per hectare per day than most other crops and requires very little input. Whereas conventional breeding techniques are difficult to apply in sweet potato due to poor seed set and sterility of flowers, genetic engineering has the potential to make a number of significant improvements to this major food crop. This potential is recognized by the numerous funding agencies that have supported projects at our institution and elsewhere.
Agronomic Improvements in Sweet Potato
Our efforts were focused initially on identifying methods to deliver foreign genes into
sweet potato (Plant Cell Reports 11:53-57). An important pre-requisite for developing
transgenic plants in any crop is the availability of tissue culture methods to regenerate
whole plants from transformed cells. Although we developed methods to produce
adventitious plants efficiently in vitro (HortScience 30:1074-1077; In Vitro - Plant
Cellular and Developmental Biology 31: 65-71), our success in developing transgenic
sweet potato owes in large measure to the development of a high-frequency somatic
embryogenesis protocol and the identification of highly regenerable cultivars (Plant Cell
Reports, in press). Genetically engineered sweet potato plants with marker genes have
now been studied under greenhouse and hydroponic conditions, and the expression
patterns of foreign genes have been characterized. Soon, we will be testing transgenic
sweet potato plants with herbicide resistance genes under field conditions.
As sweet potato plays a critical role in the diet of children in many developing countries in Africa, Asia and South Pacific, an improvement in the protein quality of this crop may have a positive impact on the health and nutrition of these people. In a study funded by NASA, we have engineered sweet potato plants with an artificial storage protein (ASP-1) gene developed by Dr. Jesse Jaynes of Demeter Biotechnologies Inc.
Several projects are underway to improve disease resistance. In one, rice chitinase and alfalfa glucanase genes have been introduced and transgenic plants will soon be tested for amino acid composition and for fungal disease resistance. We are also attempting to develop sweet potatoes with resistance to feathery mottle virus using the virus coat protein genes in collaboration with Dr. Roger Beachy of Scripps Research Institute.
In order to express the disease and pest resistance genes in a tissue-specific manner, we have cloned many genes from the periderm of sweet potato roots. The goal is to identify regulatory regions from such genes and employ these promoters to preferentially express certain resistance genes in the skin of storage roots.
Industrial and Pharmaceutical Applications
Sweet potato has very high biomass output and is grown widely, giving it considerable
potential for inexpensive mass production of novel compounds through genetic
engineering. In collaboration with Dr. Henry Daniell of Auburn University, we are
developing and testing sweet potato plants producing biopolymers. These
protein-based polymers have extensive medical applications and because of their rapid
biodegradability, are very useful in the renewable production of
environmentally-friendly plastics.
The most exciting plant biotechnology development in the past year is the production of oral vaccines in plants by expressing antigens from human pathogens. Employing the genes obtained from Dr. Charles Arntzen of Boyce Thompson Plant Research Institute, we are developing transgenic sweet potato, peanut and muskmelon plants to produce edible vaccines against the diarrhea caused by E. coli and cholera pathogens. Similarly, oral vaccine against rabies is also under development in these crops in a collaborative effort with Dr. Peter McGarvey and Dr. Hilary Koprowski of Thomas Jefferson Institute.
Tools and Techniques
High expression and appropriate targeting of foreign proteins are necessary to ensure
the success of edible vaccine production in plants. To this end, we are testing various
new promoters, enhancers and signal sequences in sweet potato using green
fluorescent protein (GFP) as a marker gene. The GFP gene, originally cloned from the
jelly fish, has high potential in plant biology research because it can be detected
without destroying the plant tissues and without using any substrates.
Polymorphic DNA markers are proving to be invaluable in plant genetic research because of their applications in the development of genetic maps, gene tagging, cloning useful genes and in studying genetic diversity. Scientists at the Center have employed the DNA amplification fingerprinting (DAF) technique to study the genetic diversity of sweet potato varieties from around the world and to fingerprint U.S. cultivars (Genome 38:938-945).
We have also identified polymorphic DNA markers for the first time in cultivated peanut using the DAF approach and the new Amplified Fragment Length Polymorphism (AFLP) technique. We are currently employing these novel markers to gain insights into genetic variability patterns and evolutionary relationships among botanical varieties of peanut obtained from various locations in South America, the home of cultivated peanut. In collaborative research with Dr. George Bruening of University of California, Davis we are employing AFLP technique to locate a marker linked to the cowpea mosaic virus resistance gene using near-isogenic lines. The AFLP markers are also being employed to investigate the genetic diversity among cowpea germplasm.
Education and Training at the Center
A major mission of the Center is to train minority students in plant biotechnology; this is
critical considering there are so few African-American or Hispanic molecular biologists.
Several minority graduate and undergraduate students are provided with research
assistantships and an opportunity to work on biotechnology projects. Similarly, high
school students are offered summer internships to work in the laboratories and
participate in research projects. This has proved very successful in enticing young
minorities to consider a career in science. To promote minority participation in plant
biotechnology, a workshop on transgenic plants is being hosted by the Center during
April 20-22, 1996.
The Center for Plant Biotechnology Research also plays an active role in training scientists and students from developing countries. Our projects have drawn visitors from Ghana, Tanzania, Zaire, Swaziland, Cote d' Ivoire, China, India, and the Dominican Republic for training in genetic engineering techniques.
C. S. Prakash, Tuskegee University
Center for Plant Biotechnology Research
prakash@acd.tusk.edu
Disappointing yields and insufficient disease resistance traits have derailed Calgene's commercialization of the Flavr Savr tomato. In its quarterly report, the company acknowledged that product quality also was in need of improvement. Production of Flavr Savr has been curtailed while new transgenic tomato varieties are being evaluated. A pending partnership with Monsanto, which seeks to buy a 49.9 per cent share of the company, is currently under review by the Justice Department. Should it be approved, Monsanto's breeding expertise is expected to strengthen efforts to develop new and improved varieties.
The news isn't all discouraging, however. Calgene appears to have come out on top in a patent dispute over the use of antisense technology. Enzo Biochem Inc. of New York had filed claims that three of its patents were infringed in the course of engineering the delayed ripening tomato. A judge in Delaware has ruled that Enzo's patents do not enable others to make use of the claimed invention, which is one of the criteria for awarding a patent, and therefore were invalid. He ruled that Calgene's patent was valid.
Pat Traynor
Seed production for hybrid plants, valued for their superior quality, yield and uniformity, depends on the prevention of self-fertilization. Use of a male sterile line as the female parent is one way to ensure out crossing. Male sterility resulting from nuclear or cytoplasmic gene expression has been identified in numerous species. The trait has been useful to breeders of many crops that normally tend to inbreed and have low levels of out crossing.
Two wild species of lettuce show genetically-based male sterility, but the recessive genes have not been useful to breeders because of pleiotropic effects and instability due to environmental conditions. Male sterility has been engineered in lettuce, as well as tobacco, oilseed rape, and other species, by introducing ribonuclease genes under the control of a tapetum-specific promoter. The lettuce, however, was not completely sterile and had abnormally shaped leaves.
A new approach, reported in Plant Science (1996; 113:113-119), shows greater promise. Collaborators at the Universities of Nottingham and Leicester introduced a beta-1,3-glucanase gene designed to be expressed in the tapetum. Previous studies at other labs had suggested that by disrupting the normal sequence of pollen development and maturation, glucanase expression could lead to male sterility. The glucanase appears to dissolve the callose wall of microspores, so that pollen grains are misshapen and nonviable. The authors note that in a plant breeding context, such transgenic lettuce plants should be amenable to cross-pollination by non-transformed plants for the production of F1 hybrid seed.
Pat Traynor
PATENTING GENETICALLY ENGINEERED ANIMALS
The patent application for Harvard's mouse model of human cancers, commonly referred to as the oncomouse, was filed in Europe more than ten years ago. The usual considerations in patent evaluation -- nonobviousness, novelty, and utility -- have become secondary to discussions of morality, and predictions are that an oncomouse patent is not likely to be granted in the immediate future.
The oncomouse patent application was refused in Europe in 1989 due primarily to an established ban on animal patenting. The application was revised to make narrower claims, and the patent was granted in 1991. This has since been repeatedly challenged, primarily by groups objecting to the judgement that benefits to humans outweigh the suffering of the animal. Currently, the patent applicant is awaiting protestors' responses to a series of possible modifications to the application. Predictions are that agreement will not likely be forthcoming and that the legal wrangling will continue into the future.
For some companies, principally those working on transgenics whose intrinsic value is in the animal itself, rather than as a source of recombinant products, this appears to be a major concern. In many countries outside Europe, patenting of animals for xenotransplantation and recombinant product production is permitted, making the European market less interesting to at least some companies. The question of patentability also affects European researchers, whose work could be inhibited by lack of access to products not covered by European patents. At least some pharmaceutical and biologics companies do a significant proportion of their research and development outside Europe in order to access animal models. It has even been suggested that some countries are falling behind the US through inability to access animal models and protocols.
Biotechnology companies may be relatively unaffected by these proceedings, in that they can usually protect inventions in some manner other than direct claims on the animal itself. Included might be claims for medical uses (transplantation of organs from transgenic animals) or processes for protein production (by the so-called "pharming" companies). It may also be possible to receive patent protection through applications to individual European countries, rather than to the central European Patent Office.
J. Glenn Songer
University of Arizona
A new pH-inducible promoter system has been characterized. The plasmid construct contains the promoter and activator coding sequences of the cad operon inserted in front of a lacZ' reporter gene. Variation in gene expression levels were observed over the pH range from 5.5 to 8, and it was shown to be a practical system for production of large amounts (up to 1.4 grams per liter, or 35% of total cellular protein) of recombinant proteins. The induction range was shown to be as high as 200 fold. Given further development and evaluation, this may prove to be another useful tool for molecular biologists seeking to improve production of recombinant proteins.
J. Glenn Songer
MORE ON RECOMBINANT PORCINE SOMATOTROPIN
The exit of Pittman-Moore from porcine somatotropin development was reportedly due, at least in part, to a desire to avoid the public fallout which has troubled the development, licensing, and sale of bovine somatotropin. Nonetheless, studies of the potential positive effects of somatotropin in pigs have continued, and a recent report presented results of testing of PST on carcass composition. Pigs were given one to three milligrams of recombinant PST daily from the time they reach 30 pounds in weight up to slaughter. Overall carcass quality and chemical composition of eleven body fractions (external fat, internal fat, breast/belly, head, neck, loin, filet, shoulder, ham, foot, edible internal organs) were evaluated. Treatment with recombinant PST caused an increase in water and protein contents, but a decrease in lipid content. The effect of sex on most characteristics was greater than the effect of PST, but significant interactions of sex and PST treatment were seen in the breast/belly cut only. External fat, breast/belly, head, neck, and feet showed the greatest PST-induced responses, but the chemical composition of the internal fat was constant. There was no recombinant PST dose effect.
J. Glenn Songer
A cDNA for chicken interferon was cloned into E. coli. A 19 kilodalton protein product of this gene accumulated in the bacteria as inclusion bodies. Purified by chromatography, its antiviral activity was approximately 108 IU per milligram. COS cells transfected with the gene produced more than 104 IU antiviral activity per milliliter of cell culture supernatant fluid. There are continuing possibilities for prophylactic or therapeutic use of recombinant interferon.
J. Glenn Songer
ALLIANCE ACTIVITY HEATS UP IN COMMERCIAL AGBIOTECH
SECTOR
The strategic alliance and acquisition activity that has been a cornerstone of the biopharmaceutical industry is also becoming the model for agricultural biotechnology. As described in the August 1995 edition of Agricultural Biotechnology Notes, many of the drivers that have led pharmaceutical and biotechnology firms to partner in recent years, including access to technology, distribution channels, and capital, are transferable to commercial agriculture. The ag-sector's growing propensity to partner has been demonstrated recently by some of the industry's major players.
Monsanto continues to establish itself as a leader in agricultural biotechnology, most recently through the acquisition of equity in the seed company DeKalb Genetics. Monsanto will pay up to $158 million to DeKalb in an agreement that includes an equity stake for Monsanto and a 10 year cross- licensing pact that has the two firms sharing revenues and royalties. The agreement does not transfer rights to any of DeKalb's proprietary inbred, hybrid or varietal seed lines. The deal does give Monsanto another valuable link in commercializing its biotechnologies in the form of access to an established seed distribution system (1). DeKalb, on the news of the Monsanto collaboration and the awarding of two key biotech patents, has seen its stock soar in recent weeks. The DeKalb deal is just the most recent in a chain of biotech-related alliances that Monsanto has developed, including its investment in Calgene, a collaboration with Ecogen to develop insect resistant plants, and an agreement with Delta and Pine Land Company regarding herbicide resistant cotton (1).
Not to be outdone, DowElanco and Mycogen Corp. announced an agreement, that when complete, will give DowElanco a 46 percent equity interest in Mycogen. As part of the agreement, DowElanco will pay $126 million to Lubrizol Corp. for its 27 percent stake in Mycogen, as well as turn over its United AgriSeeds business and $26.4 million to Mycogen in exchange for 4.5 million shares of Mycogen common stock (2). The alliance better positions both companies to more aggressively compete in the growing agbiotech market.
In another significant deal, Empresas La Moderna of Mexico paid $10 million to take a 70 percent equity position in DNA Plant Technology (DNAP). DNAP will combine with Moderna's Bionova produce unit to create a new company, the DNAP Holding Corp., which will receive $30 million in research contracts from Empresas La Moderna over the next 10 years (2).
In addition to setting up alliances with agbiotech firms, major agriculture companies are also looking to biopharmaceutical firms to access new technologies. Pioneer Hi-Bred International announced a five year agreement with Human Genome Sciences for corn genomic research and development. DowElanco is looking to Chiron Corp. to access combinatorial chemistry for screening novel agrochemicals (1).
Beyond directly enriching the companies involved, alliances that include large multinational agricultural companies and established biopharmaceutical firms indirectly benefit the entire agricultural biotechnology industry by enhancing its credibility which, in turn, may heighten investor interest and boost capital flow into the sector.
References:
William O. Bullock
Institute for Biotechnology Information, LLC
Research Triangle Park, NC
BIOSCI - A NETWORK FOR BIOLOGISTS
Whether you are a scientist, teacher or a student in biotechnology, every day you are faced with a question or situation that could best be answered by other biotechnologists. What type of thermal cycler should I buy that fits my need and the budget? Where can I get the new plasmid with the green fluorescent protein gene? How do you dissolve thidiazuron? How I can contact Dr. Joan Doe by email to ask her about the DNA isolation protocol from herbarium samples? Where can I find out about recent job listings or research assistantships? Is there software that can help me analyze gel images? Where can I get details of NIH grant programs? What papers were published in the February issue of "Nucleic Acids Research?".
A one-stop answer to many of the information needs of a practicing biotechnologist is the BIOSCI network on the Internet, an electronic communication forum. More than 30,000 biologists use this network of newsgroups and thus it can be a valuable source of information. BIOSCI had its origins in 1984 as a bulletin board system on the BIONET National Computer Resource for Molecular Biology at IntelliGenetics Inc. The network, coordinated by Dr. David Kristofferson (biosci-help@net.bio.net), now has almost a hundred discussion groups. In Methods, the most popular group, users discuss protocols and methods, reagents and various techniques in molecular biology. Other groups include Arabidopsis, molecular evolution, RAPD (for DNA markers), photosynthesis, Drosophila, yeast, and women-in-biology. The "Bio-Journals" section posts the table of contents of close to 75 journals. There are lists for those seeking employment and a separate one for those advertising positions. There is also a newsgroup for information on funding agencies such as NIH and NSF. The database of BIOSCI members is a useful means of locating email addresses, phone and fax numbers of many biotechnologists around the world.
Generally, members offer very helpful information and advice in response to postings, and thus the use of BIOSCI newsgroups can be very rewarding. Further, all the postings are archived and can be easily searched using appropriate key words. In fact, it is suggested that users first search in the archives before posting specific questions so as to pick up informative discussion threads on many topics.
There are three ways of reaching the BIOSCI network. The best is on the world wide web (http://www.bio.net because of the graphical interface and hyperlinks. The BIOSCI newsgroups can also be accessed through the USENET and subscribed through email. Send an email message to biosci@net.bio.net for detailed information on how to access BIOSCI through USENET and email.
C. S. Prakash, Tuskegee University
Center for Plant Biotechnology Research
prakash@acd.tusk.edu
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.
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