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November 1997 | |
NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
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November 1997 | |
NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
PLANT BIOLOGISTS CONVERGE IN SINGAPORE
Forest fires raging over thousands of acres in neighboring Indonesia cast a dense smog over
Singapore. But the bad air did not dampen the enthusiasm of nearly two thousand plant molecular
biologists from 79 countries who gathered in this island-nation to attend the 5th International
Congress of Plant Molecular Biology, September 21-27, 1997.
In her opening address, Adrienne Clarke (Australia), president of the International Society for Plant Molecular Biology, said that plant molecular biology is expected to be increasingly important in agricultural research to feed the expanding world population. She announced that the Society is developing literature addressing issues on the regulation of genetically modified food products and will initiate an outreach program for developing countries.
The Singapore conference hosted plenary lectures from 16 invited scientists who spoke on various issues such as plant development, genomics and disease resistance. Thirty-two concurrent sessions featured 350 papers on a wide spectrum of topics including biotic and abiotic stress, gene silencing, flowering, growth regulators, cell cycle, genome research, product development for agriculture, forestry products, crop transformation and tropical crop biotechnology.
Special workshops were held on gene transfer technology, biocomputing, Agrobacterium, and gene nomenclature. Nearly 1100 posters were also presented. As more than half the participants were from Asia, rice figured in many presentations. Many of these scientists had also attended a conference sponsored by the Rockefeller Foundation on rice biotechnology held the previous week in neighboring Malaysia.
According to Chris Somerville (USA) who described international efforts of the Arabidopsis genome project, plant biology is in the midst of a paradigm shift towards a more interactive approach to research similar to those undertaken in physics. He predicted that plant genome scientists would increasingly employ DNA chips in their research, and functional analysis of the genome through such approaches as gene knockout will be employed.
Caroline Dean (UK) announced the cloning of a gene for early flowering from Arabidopsis, while Barbara Baker (USA) illustrated how induced mutations in a tobacco mosaic virus resistance gene are contributing to our understanding of disease resistance in plants. Using the transposable elements identified by the late Barbara McClintock, Venkatesan Sundaresan (Singapore) has developed a novel gene trapping system for locating new genes in plants.
To meet the challenges of increasing food production and food security in developing countries, all available tools must be employed and plant molecular biology research will have a larger stake in future agricultural research, according to Ingo Potrykus (Switzerland). His research is focused on developing improved varieties of rice and cassava with resistance to diseases and pests, and with enhanced food quality attributes. Swapan Datta (Philippines) shared some impressive results from transgenic rice that show resistance to diseases and with increased tolerance to submergence.
Farmers growing transgenic plants in the US are highly positive and receptive to this new technology, announced Dilip Shah (USA) of Monsanto Company. Herbicide-tolerant soybean was grown on 9 million acres in the US during 1997, accounting for 25% of the soybean acreage, while Bt cotton and Bt corn were grown on 2 million and 3 million acres, respectively. Shah reported that insect-resistant Bt cotton showed only 2% damage while the unsprayed regular cotton crop had 25% insect damage. Even with pesticide sprays, the nonengineered cotton showed 12% damage. Farmers reaped a $33 advantage per acre with the Bt cotton, according to Shah. Monsanto is now testing many novel insecticidal genes including cholesterol oxidase which appears promising against the cotton boll weevil.
Hundreds of posters described various creative approaches to develop new agricultural products and processes. A few with esoteric applications that caught my attention included the engineering of plants to produce drugs against helminth infection, canola producing recombinant fish growth hormone, developing a rice variety rich in iron, engineering antibody genes to mediate resistance to plant pathogens, use of an ethanol-sensitive promoter to control plants gene expression through alcohol sprays, and plants engineered to reduce air pollution.
Caroline Dean (UK) has been elected the next president of this Society which will host its next congress in the year 2000 at Quebec City, Canada.
C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu
SYMPOSIUM ON ANIMAL BIOTECHNOLOGY
An international symposium on Genetically Engineering and Cloning Animals: Science, Society
and Industry, will be held next summer in Park City/Deer Valley, Utah. The conference,
scheduled for June 21-23, 1998, is sponsored by the Utah State University Biotechnology Center.
The program will address a broad range of issues associated with generating transgenic animals, including the status of basic science, public policy, FDA regulations, patent issues, and economics. Topics are grouped in three areas:
The Symposium website ( http://www.usu.edu/~biotech/tranhome.html) is still under construction but some information is posted. For further information, contact Nancy Ashcroft, Conference Secretary at 435-797-2753, fax 435-797-2766; email: nancya@cscfs1.usu.edu
PLANT RESEARCH NEWS
ANTIVIRAL PROTEIN CONFERS INCREASED FUNGAL RESISTANCE
Plants employ a variety of defense mechanisms to ward off invading pathogens, often
synthesizing chemical toxins in order to kill either the pathogen or the plant's own cells in
the area of invasion. Among such toxins produced by many plant species are ribosome-inactivating
proteins (RIPs), which destroy the function of ribosomes and thus stop protein
synthesis. RIPs were first found to be effective in inhibiting viruses and have received
increasing attention from scientists interested in using them to engineer pathogen resistance
in plants. The substrate specificity of RIPs can vary considerably, with some showing
preference for mammalian or fungal ribosomes, while others will attack both eukaryotic and
prokaryotic ribosomes equally. One such broad-spectrum RIP is the pokeweed antiviral protein
(PAP). When the gene for this protein was expressed in transgenic tobacco it increased the
plant's resistance to virus, but was also toxic to the plant itself, resulting in slower growth
and leaf chlorosis and lesions.
In the October issue of Nature Biotechnology, Nilgun Tumer and colleagues report intriguing results of experiments with transgenic tobacco plants expressing non-toxic mutants of PAP (1). They made mutations to the PAP gene that caused the enzyme to lose its activity and then expressed the new genes under the control of the constitutive cauliflower mosaic virus promoter.
One gene construct had a point mutation that altered the active site of the enzyme while a second construct had a mutation that caused the protein to be shortened by 25 amino acids at the carboxyl end. Since both of the resulting enzymes were nonfunctional, they were not toxic to the transgenic plants, which grew identically to non-transformed plants. Nevertheless, the transgenic plants had increased resistance to the fungus Rhizoctinia solani as well as to viral attack. One line expressing the carboxyl end deletion showed slower onset of injury symptoms following fungal inoculation and a 63% reduction in mortality as compared to wild type control plants. This is an interesting development because even when functional, PAP has no direct toxicity to fungus in culture.
These results raise an interesting question for plant pathology: How can the nonfunctional protein enhance plant resistance despite loss of its enzymatic activity? Since the non-toxic enzyme was not affecting the fungus itself, it must be acting indirectly by modifying the plant defense responses. Examination of some aspects of the plant defense system support this hypothesis. The increase in fungal resistance was correlated with higher continuous expression of pathogenesis related (PR) proteins, which are important in the hypersensitive response. Expression of both mutant and non-mutant PAP genes increased levels of PR proteins, though the active-site mutant gene was not as effective in this respect as the carboxyl end mutant. Levels of salicylic acid, an important signaling molecule in plant defense responses involving PR proteins, were not altered by the presence of any PAP constructs, suggesting that PAP stimulates the plant defense signal transduction pathway downstream from the point of salicylic acid involvement. The PR protein induction appears to be a specific response because mutant PAP did not affect plant ribosomes, so increased resistance was apparently not the result of general cell stress caused by inhibition of protein synthesis.
While it remains unclear exactly how pokeweed antiviral protein triggers increased plant defense to pathogens, this research demonstrates that the toxicity of PAP to plant ribosomes is not essential for its function in defense. Somehow, the expression of even a non-functional PAP acts as an alarm that causes the plant to be constantly prepared for pathogen attack. This offers hope that similar mutant protein constructs can be used to engineer disease resistance into other crop plants without the detrimental effects of the wild type PAP.
Reference
1. Zoubenko, O., F. Uckun, Y. Hur, I. Chet, and N. Tumer. 1997. Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants. Nature Biotechnology 15:992-996.
Jim Westwood
International Research and Development
Virginia Tech
westwood@vt.edu
NEW AND IMPROVED PLASMIDS AVAILABLE
Plasmid vectors are essential for developing transgenic plants. These tiny DNA circles are
the unsung workhorses of biotechnology, helping scientists shuttle genes into plant cells that
are then regenerated into transgenic plants. Current plasmid vectors have many limitations, and
thus scientists at the Center for the Application of Molecular Biology to International
Agriculture (CAMBIA) in Canberra, Australia have developed improved vectors which they are
making freely available to academic researchers. Richard Jefferson, who is the head of CAMBIA,
says that the new CAMBIA vectors are highly efficient and useful in developing transgenic
plants and are extremely versatile for plant molecular biology research.
Jefferson started CAMBIA to develop "enabling technologies" of relevance to international agriculture. The GUS gene system, which he developed in the mid-eighties at Cambridge and made widely available to researchers even before publication, has made a tremendous impact on plant molecular biology. Transformed plant cells expressing the GUS gene appear dark blue in color when stained with a substrate and thus can be easily identified.
The new upgraded CAMBIA vectors consist of 18 plasmids and have several modular features: choice of selection in plants (hygromycin or phosphinothricin resistance), choice of selection in bacteria (chloramphenicol or kanamycin resistance), multiple cloning sites close to the right border (ensuring no loss of the introduced gene), blue/white screening in E. coli, presence of an intron sequence (to suppress GUS expression in the bacteria), high copy number plasmid replication, Kozak consensus sequence for improved expression, minimal extraneous DNA sequences (such as spurious AG or terminator sequences) and stability of plasmids under non-selection conditions.
Further, a protein of interest can be fused to the GUS protein and the new protein purified with a single step because of the hexahistidine tail engineered at the carboxyl end of GUS. A promoter-less vector and a reporter gene without start codon are also available. All vectors have been tested successfully with tobacco and rice using particle bombardment and Agrobacterium transformation methodologies.
Recently green fluorescent protein (GFP) from jellyfish has emerged as a viable alternative to the GUS gene, as GFP could be visualized in plant cells non-destructively. Jefferson cautions, however, that GFP has several caveats such as its unsuitability for field use, stochiometric detection which limits sensitivity, need for use of expensive microscopy and the photo toxicity of the protein. Nonetheless, he is also offering a codon-modified GFP (developed by Jim Hasseloff of UK) in the CAMBIA vector along with GUS-GFP (or GFP-GUS) gene fusions, thus providing scientists with a single system with features of both reporter genes.
The original GUS gene was isolated from E. coli and has certain drawbacks. To improve the plasmid vector system further, CAMBIA scientists have cloned an improved GUS gene from another newly described bacteria, Bacillus oz, and call this gene "BoGUS". The BoGUS gene is a major improvement because it is secretory in nature. It is also more robust with very high thermal stability, according to Jefferson. BoGUS is not inhibited by its end products or detergents and thus is active even in high concentrations of salt and organic solvents. This gene is also being further improved through codon optimization.
Jefferson visualizes that the new BoGUS will facilitate a non-destructive and non-disruptive assay of gene expression and will facilitate the development of transgenic plants without antibiotic selection or tissue culture. The new gene will require cheaper substrates and may have other applications such as targeted cell death to cause male sterility through special substrates. The new BoGUS gene will be made available to researchers soon.
Academic researchers can obtain the CAMBIA vectors at no cost while commercial institutions are required to license them. For more information, send email to: cambia@cambia.org.au. Richard Jefferson can be reached at raj@cambia.org.au
C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu
ANIMAL RESEARCH NEWS
MOLECULAR BASIS FOR BEEFIER BEEF
Belgian Blue cattle and a number of other cattle breeds display an increase in muscle
development that is commonly known as double muscling. These breeds typically show a 20%
increase in muscle mass due to an increase in the number of muscle fibers. Double muscling,
however, also causes serious problems such as difficult calving, low viability of double
muscled calves at birth, and poor milk production. In spite of these reproductive problems, the
double muscling trait has been of interest to beef producers.
In Belgian Blues, the gene that is responsible for double muscling appears to be inherited as an autosomal, recessive gene. This suggests that the animals contain two copies of a mutated gene involved in muscle development, however the molecular basis for the double muscling phenotype until recently had not been identified.
An international group of researchers from Belgium, France, Spain, and Germany report in the September issue of Nature Genetics that the double muscling phenotype in Belgian Blue cattle is due to a mutation in the bovine myostatin gene. Myostatin is a member of a large family of growth and differentiation factors that play important roles in regulating embryonic development and tissue growth.
Previous studies from a group at Johns Hopkins University demonstrated that in mice myostatin is specifically expressed in adult skeletal muscle and that deletion of both copies of the myostatin gene resulted in mutant mice with an overall increase in muscle mass. Some individual muscles showed a two- to three-fold increase in weight. These results suggest that myostatin acts as a negative regulator of skeletal muscle growth and that deletion of this gene causes an increase in muscle mass.
In Belgian Blue cattle, the mutated myostatin gene had a deletion of 11 base pairs in the coding region. As expected, both copies of the gene were defective. The status of the myostatin gene was also examined in the double-muscled Asturiana and Maine-Anjou cattle breeds. The Asturiana cattle, like the Belgian Blues, contained two copies of the defective myostatin gene, whereas the Maine-Anjou cattle surprisingly contained two copies of the normal myostatin gene. Thus, in not all breeds of cattle is double muscling caused by a mutation in the myostatin gene.
The identification of the molecular basis that causes double muscling in two breeds of cattle is significant because it will allow the development of a diagnostic test for the trait. This would aid in the selection for or against the trait, because carrier or double-muscled animals can be more accurately identified.
References
Grobert, L. et al. (1997). A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nature Genet. 17:71-74.
McPherron, A.C., Lawler, A.M., and Lee, S.-J. (1997). Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83-90.
Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu
TRANSGENIC PIGS TO THE RESCUE
In the October issue of Nature Biotechnology, two papers exemplify the exciting medical
applications of transgenic livestock, in these cases transgenic pigs. One report describes the
production of the human blood coagulation factor VIII in pig milk (1), while the other
describes the development of transgenic pigs as models for human retinitis pigmentosa, a
degenerative disease of the retina (2).
Approximately one in ten thousand human males suffers from hemophilia A, which is an X-linked disease caused by the lack of functional blood coagulation factor VIII. Preparations of human factor VIII from plasma have been successfully used as a treatment for hemophilia A. However, due to contamination of the blood supply with blood-borne viruses, in particular human immunodeficiency virus (HIV), greater than half of the treated hemophiliac patients from 1977 to 1985 were infected with HIV. This tragic result reemphasized the need for extremely rigorous testing or development of alternative sources of blood-derived products. Because factor VIII is a large protein that requires a number of protein modifications for biological activity, its production in bacteria is not feasible.
The mammary gland is an ideal organ to target for the synthesis of a foreign protein because it is designed for large volume production of a protein-rich solution, i.e., milk. In addition, milk can be collected non-invasively with minimal stress to the animal. Although dairy cattle are the preferred animals for production of foreign proteins because of the large volume of milk produced, technical factors such as generation interval and number of offspring make transgenic pigs a more logical choice. In the Nature Biotechnology report, human factor VIII was produced at a concentration of 2.7 milligrams per liter of milk. Although this level of expression is low, the report is significant because it demonstrates the feasibility of producing a large and complex protein in a biologically active form in the milk of a transgenic animal. Further modifications of the factor VIII transgene will undoubtedly lead to higher levels of expression.
In the second report, transgenic pigs were developed as an animal model of a human eye disease. The eye is composed of rod and cone photoreceptor cells, which are responsible for vision in low light and color vision in bright light, respectively. Patients with retinitis pigmentosa typically develop night blindness early in life due to the loss of rod cells. The remaining cones are slowly lost over time leading to blindness. To mimic these symptoms in humans, transgenic pigs were developed which contain a defective rhodopsin gene. Rhodopsin is the photosensitive molecule in rods that responds to light. In these pigs there is an early and severe loss of rods and a gradual loss of cones similar to that observed in the human disease. Due to the similarity in eye size, these pigs provide an invaluable animal model for testing surgical procedures or drug treatments for individuals suffering from retinitis pigmentosa.
These are just two of the exciting applications of transgenic pigs. A third application that is currently in development is the use of transgenic pigs as organ donors. Clearly, the status of pigs in old MacDonald's medical pharm has been raised a number of notches from mud and slop wallower to challenger for the title of barnyard king.
References
1. Paleyanda, RK et al. (1997). Transgenic pigs produce functional human factor VIII in milk. Nature Biotechnology 15:971-975.
2. Petters, RM. et al. (1997). Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa. Nature Biotechnology 15: 965-970.
Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu
INDUSTRY NEWS
PIONEER HI-BRED CONTINUES TO MAKE DEALS
Leading agricultural biotechnology corporation Pioneer Hi-Bred International has been active
on the business front over the past few months. In August, Pioneer announced the formation of
an alliance and joint venture with DuPont to speed the discovery, development and delivery of
new technologies and crops in the coming years. As part of the agreement, DuPont will invest
$1.7 billion in Pioneer, ultimately owning 20 percent of its stock for $104 per share and two
seats on Pioneer's board of directors. The $104 per share is at a premium to Pioneer's market
price of $93 per share (as of the writing of this article). The vision behind the alliance is
to create synergy by bringing together DuPont's capabilities in materials sciences and
biotechnology with Pioneer's global strength in corn and oilseed genetics to nurture the
genesis of new products.
The alliance creates one of the world's largest private agricultural research and development collaborations, with the companies combined investing over $400 million in agricultural research in the next year. A portion of the budgets will go to directly support the new joint venture through collaborative research in genetic modification of corn, soybeans, and other oilseeds to improve their oil, protein, and carbohydrate composition. The equally owned joint venture, Optimum Quality Grains, will work to bring newly developed products to the market.
DuPont anticipates taking a one-time, non-cash charge to earnings in relation to the deal, funding the transaction with cash flow from operations and debt, as needed. The charge will be taken as a write-off assigned to in-process research and development, and is not expected to exceed $1 billion.
Pioneer is using a portion of the proceeds to buy back some of its own stock. In late October, the company announced that it anticipated purchasing approximately 16.4 million shares at a price within an estimated range of $92.50 to $94 per share in a Dutch auction self tender offer. The company's stock has risen from a past year low in the $50s to its current value per share in the $90s. Part of the reason for the markets support of the stock is the company's overall performance.
Pioneer's earnings for fiscal year 1997 rose 9 percent to a record $243 million, on record sales of $1.784 billion. Pioneer achieved a return on ending equity (i.e., ratio of profit to shareholder's equity (stock)) of 21 percent, representing the forth consecutive year in which the company has achieved its goal of at least 20 percent ROE. Pioneer management anticipates an even better year in 1998, announcing projected earnings for the next year in the range of $3.20-$3.60 per share, as compared to $2.95 per share in fiscal 1997.
Pioneer has recently done a number of other deals related to biotechnology (see previous NBIAP News Reports). The company's healthy financial position and growth expectations, in addition to major deals like the DuPont alliance, provide strong validation for the future of biotechnology as a cornerstone in commercial agriculture.
Reference
Pioneer Hi-Bred web site, www.pioneer.com, 1997.
William O. Bullock
Institute for Biotechnology Information
Research Triangle Park, NC
wbullock@mindspring.com
http://www.biotechinfo.com
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