 |
September 1997 | |
NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
|
September 1997 | |
NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
New DNA Chip Technologies Impact Agbiotech Research
Commercialized Crops Spawn Secondary Biotech Products
No Small Potatoes: Yeast Gene Dramatically Alters Tuber Size
Steady Progress in Tree Research
Cattle Cloning Advances with Introduction of 'Gene'
Study Shows Sizable Potential Impact of Agbiotech in Europe
NEW DNA CHIP TECHNOLOGIES IMPACT AGBIOTECH RESEARCH
Compared to genome scientists working with crops and livestock, human genome researchers have
some major advantages: better funding, more visibility, good corporate support, a large market and
a critical mass of scientists whose effort is focused on a single system. The combination of these
factors has driven the development of improved technologies at breakneck speeds. Now, the high
powered tools being developed to analyze the human genome are making inroads into agricultural
biotechnology. Such tools are essential for genome researchers to move beyond sequencing and into
the next phase of research where they conduct enormously large scale gene discovery surveys and
gene expression analyses.
DNA chips represent such a "massively parallel" genomic technology (1). They facilitate high throughput analysis of thousands of genes simultaneously, and are thus potentially very powerful tools for gaining insight into the complexities of gene expression, detecting genetic variation, making new gene discoveries, fingerprinting cultivars and developing new diagnostic tools.
Two types of DNA chips now available are based on the principle of hybridization in which nucleotides on complementary nucleic acid strands recognize each other through base pairing. Only tiny amounts of DNA and chemical reagents are needed and sample preparation effort is minimal.
To produce a 'synthesized' DNA chip, a huge number of oligonucleotide probes are synthesized directly on a glass surface or silicon wafer using a process called photolithography (1). More than 400,000 such probes can be placed on a single chip measuring 1.28 cm X 1.28 cm. Next, a fluorescent labeled target nucleic sequence is hybridized to the probes on the chip, and the resulting fluorescent image is scanned by a laser beam and analyzed by a computer. The intensity of fluorescent light varies with the strength of the hybridization thus providing a quantitative 'snapshot' of the gene expression. Even very rare mRNA species (1 in 300,000) can be detected by this approach thus making it possible to detect 'hard-to-find' genes. The process is automated and now commercially available.
Affymetrix, a company located in Santa Clara, California manufactures five to ten thousand DNA chips per month and is targeting applications such as screening for human immunodeficiency virus or genes associated with cancer, and gene resequencing (1). The potential for this technology is enormous and 'only limited by imagination' according to Stephen Fodor, CEO of Affymetrix. The company already is supplying Pioneer Hi Bred with custom DNA chips for monitoring corn gene expression. Affymetrix has established programs where academic scientists can use company facilities at a reduced price and they are also setting up 'user centers' at selected universities.
A related but less complex technology called DNA microarray or 'spotted' DNA chips involves precisely spotting very small droplets of genomic or cDNA clones or PCR samples on a microscope slide (2). The process uses a robotic device with a print head bearing fine tweezers that work like fountain pens to draw up DNA samples from a 96-well plate and spot tiny amounts on a slide. Up to 10,000 individual clones can be spotted in a dense array within one square centimeter on a glass slide. After hybridization with a fluorescent target mRNA, signals are detected by a custom scanner. A description of DNA microarray technology, including short videos of the arrayer and scanner in action and a picture of the complete yeast genome on a single chip, can be viewed at http://cmgm.stanford.edu/pbrown/array.html.
Shauna Somerville at the Carnegie Institution of Washington in Stanford, CA says that DNA chip technology would play an important role in plant biology in the future, but right now 'synthetic' DNA chips are too expensive and limited in applications to agricultural biotechnology because their use requires a prior knowledge of gene sequences. Such information in crop plants and livestock is still relatively limited. A spokesperson from Affymetrix maintains, however, that the commercial costs of the two technologies are comparable.
Somerville is collaborating with biochemists Pat Brown, Ron Davis and Mark Schena at Stanford University in using the DNA microarray (spotted DNA chips) technology to understand how plants respond to pathogen infection. In a preliminary study, her group observed for the first time that the model plant Arabidopsis turns on many genes in response to an infection by powdery mildew pathogen and turns off many other genes (3). While some of the activated sequences are known defense-related genes from other crop plants, a few were not known earlier to be involved in plant-pathogen interactions.
Somerville believes that once the Arabidopsis genome is completely sequenced, the greatest challenge facing plant biologists will be to determine the function of all genes uncovered by sequencing. Both the synthetic and spotted versions of the DNA chip technology will be important for gene characterization in the post-genome sequencing era.
These technologies permit scientists to conduct large scale surveys of gene expression in crop plants, thus adding to our knowledge of how plants develop over time or respond to various environmental stimuli. The new techniques will be especially useful in gaining an integrated view of how multiple genes are expressed in a coordinated manner.
Although many fundamental plant processes can be studied in Arabidopsis, some problems can only be studied in crop plants. Large scale sequencing projects for important crop plants such as corn or potato, using expressed sequence tags coupled with DNA chip technology, have the potential to dramatically enhance our knowledge of how complex agronomic traits such as yield or adaptation to stresses (e.g., salt and drought stress, temperature extremes) are controlled, says Somerville. The increased knowledge can provide powerful tools to redesign crop plants to be more productive under extreme environments.
Currently, an initiative to fund a major crop genome effort is being discussed in the halls of the U. S. Congress. If passed, the initiative will be a tremendous boost for plant scientists anxious to employ modern technologies in their research to improve the productivity of organisms which collectively make up dinner on the dining table.
References
1. Fodor, S. P. A. 1997. Massively parallel genomics. Science 277:393-394
2. Shalon, D. Et al. 1996. A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res. 6:639-645.
3. Schena, M. et al. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467-470.
C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu
COMMERCIALIZED CROPS SPAWN SECONDARY BIOTECH PRODUCTS
Hundreds of millions of dollars have been spent on engineering of crops to induce pest resistance and
herbicide tolerance. These transgenic products are being enthusiastically embraced by growers
because of the significantly reduced expense and effort to combat insect damage and herbicide
exposure. The full potential of this technology, however, can only be realized if accurate and easy
verification methods for genetic traits are available.
Strategic Diagnostics, Inc. (SDI, Newark, DE) has stepped into this market with immunoassay testing technology that is ideally suited for transgene verification. SDI's first product, GeneCheck® B.t.k., was developed under contract with Monsanto to detect the insect toxin produced by Bacillus thuringiensis kurstaki. This toxin is produced in a variety of crops engineered for insect resistance, including corn, cotton and soybeans.
The proprietary GeneCheck test is a 10 minute, field-portable immunoassay with a non-enzymatic visual detection method that uses gold particles. The test kit consists of only a disposable microcentrifuge tube, a few drops of buffer, a disposable micro tissue grinder and a detection strip similar to those used for home pregnancy tests. For only a dollar or two apiece, hundreds or even thousands of plants can be screened to verify the presence of a transgene product.
Monsanto's Bollgard insect-resistant cotton has been described as the most financially successful new agricultural product introduction in the U.S. The SDI test is a crucial part of the quality assurance checks employed by seed supplier Delta and Pine Land Company (Scott, MS) and others during seed propagation and quality control of seed production. About a million SDI B.t.k. tests were used for this process in 1996. Other potential applications could include monitoring of seed lots to prevent mixing of seed and spot-checking growers' fields to verify compliance with licensing requirements for refugia.
According to James Stave, Vice President for R&D, SDI is actively partnering with leading developers of transgenic crops to produce similar immunoassay tests for the detection of other transgene products.
Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
PLANT RESEARCH NEWS
NO SMALL POTATOES: YEAST GENE DRAMATICALLY ALTERS TUBER SIZE
Plant physiologists have a somewhat unappetizing name for plant parts that store the carbohydrates
we love to eat. The apple fruit, wheat grain, peanut seed and potato tuber are called 'sinks' because
these organs import and store carbohydrates produced by photosynthetically active organs such as
leaves. Farmers and consumers alike pay a lot of attention to sinks, and thus scientists have long
struggled to improve the quantity and quality of storage organs in plants.
Fundamental insights into how plants transport and store food reserves and how sugar metabolism affects the size of storage organs such as potato tubers are critical to improving crop productivity. Lothar Willmitzer's group of Max Planck Institute in Germany have pioneered the study of potato starch and sugar metabolism for more than a decade. A recent report from Willmitzer and his associates gets us closer to understanding the link between metabolic pathways and tuber size. They showed that an invertase enzyme from yeast could increase potato tuber weight more than three-fold when targeted to extracellular spaces (apoplastic expression). Interestingly, the same gene product targeted within the cell (cytosolic expression) reduces the potato tuber weight by almost one third (1).
Sucrose is synthesized in leaves and transported to the tuber where it is distributed by specialized cells called sieve elements. Sucrose is then broken down to the simple sugars glucose and fructose, usually by the enzyme sucrose synthase and sometimes by invertase. The German group developed two types of transgenic potato plants both expressing the invertase gene (suc2 from brewer's yeast) in the tuber with the help of a patatin promoter. In one type, expression of the invertase was localized in the cytosol, while in another set of plants invertase expression was targeted to extracellular spaces (apoplastic) using a signal peptide from the potato proteinase inhibitor II gene.
When transgenic potato plants were tested for two seasons in the greenhouse, no differences in the growth and development of plants were observed above ground. It was below ground where surprising results were apparent, as there were clear differences in the tuber size and number. In a transgenic line where the yeast gene was targeted to extracellular spaces, tubers weighed an average of 57 grams each compared to 18 grams in the non-engineered control plants.
Such increases, however, were accompanied by a proportionate decrease in the number of tubers produced per plant. In contrast, in plants where the invertase was expressed within the cytosol, the tuber weight ranged from 6 to 12 grams while the tuber number was nearly doubled (from 8 per plant to 18). A subsequent field trial confirmed the trend although the changes were not as dramatic. The cytosolic expression of yeast invertase clearly brought down potato yield and tuber size, while the apoplastic expression increased both, at least under greenhouse conditions.
It is not known yet why potato tuber size changes depending on where the invertase is localized, but the authors offer two theories. One possibility is that breakdown of sucrose by invertase localized in the extracellular spaces may lower the turgor pressure within the cells, leading the tuber to unload sucrose through the plasmodesmata. When invertase is expressed in the cytosol, the reverse happens as increased cell turgor would reduce such unloading.
Alternatively, glucose accumulated in the extracellular spaces of the apoplastic lines, may act as a signal to trigger cell division leading to tuber enlargement. The glucose accumulated within the cells of cytosolic lines may not act as such a signal. Whatever the reason, the Nature Biotechnology paper clearly shows that location of breakdown of sugar within the sink can impact the potato tuber size, and this has clear implications in improving the potato crop.
Reference
1. Sonnewald, U. et al. 1997. Increased potato tuber size resulting from apoplastic expression of a yeast invertase. Nature Biotechnology 15:794-797.
C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu
STEADY PROGRESS IN TREE RESEARCH
The latest news from the Tree Genetic Engineering Research Cooperative (TGERC) is compiled in
their 1996-97 Annual Report. TGERC is a consortium of forest industry companies, Oregon State
University, and the U.S. Department of Energy Biofuels Feedstock Development Program. The
cooperative is engaged in research, technology transfer, and education to facilitate use of genetically
engineered trees in plantation culture.
All TGERC research is oriented toward three goals closely related to commercialization: (1) efficiency of gene transfer and transgene expression; (2) large-scale field testing; and (3) environmental safety of engineered plantations. Some of the highlights from the report are described below. For more information, visit the TGERC website ( http://www.cof.orst.edu/coops/tgerc/) or contact Steve Strauss at 541-737-6578, strauss@fsl.orst.edu.
Transformation
Two areas of transformation research are yielding good results. First, a simple protocol developed
for transforming diploid and triploid Populus trichocarpa X P. deltoides (TD) hybrids will speed
progress towards field testing commercial hybrids containing insect resistance, herbicide resistance,
and sterility genes.
The second area involves the use of matrix attachment regions (MARs) to improve the rate of transformation. MARs are segments of DNA that interact with chromatin protein. Preliminary evidence suggests that they may be of considerable value in enhancing the level and uniformity of transgene expression in poplars. Flanking the GUS reporter gene with a MAR element from a tobacco genomic clone doubled expression and reduced variation among greenhouse-grown lines. Interestingly, MARs had little effect on GUS gene expression in tobacco.
Insect Resistance
Research on resistance to insect damage has two main goals: the development and testing of
transgenic trees with Bt toxin genes, and studies of genetic and ecological factors important to
resistance management. Both areas focus on resistance to defoliating insects, particularly the
cottonwood leaf beetle (CLB).
A Bt gene that exhibits high toxicity to CLB has been transformed into poplar hybrids. The cry3A gene, provided by Mycogen Corporation, was flanked with MARs and introduced by a standard Agrobacterium protocol. Preliminary tests of feeding rates and larval mortality were highly encouraging, and more extensive screening of greenhouse-grown plants is under way. Field trials are being planned for 1998.
Over time, pest populations exposed to insecticidal compounds including Bt toxins develop resistance. Cottonwood leaf beetle poses a substantial risk of becoming resistant because it undergoes many generations per year and has been shown to have the genetic potential to develop strong resistance. Controlled crosses between susceptible wild populations and Bt-resistant laboratory populations are being used to study the genetic basis of resistance in CLB.
The picture that is emerging indicates that resistance is at least partially recessive, and there is some evidence suggesting the existence of multiple resistance loci. These genetic studies are being continued to help identify the most effective strategies fro preventing beetle populations from developing Bt resistance. The genetic map of CLB that will come from this work may lead to molecular markers useful for monitoring resistance development in the field.
Genetic Risk Analysis
Factors that can contribute to the movement of transgenes include fertility of the hybrid clones used
in the Northwest, potential gene flow by pollen and seed from existing hybrid plantations, and
establishment and competitiveness of hybrid progeny relative to wild progeny.
Ongoing studies are assessing the fertility of TD hybrid clones in the hope that these triploids are sufficiently sterile to provide adequate gene containment for rotation-length field tests of transgenic trees. After controlled crosses, pollen viability, seed production, seed germination, and progeny fitness are being evaluated
Information on gene flow from existing (non-transgenic) poplar plantations will provide data for simulation studies of potential gene movement. Progeny of wild females growing near hybrid plantations are being screened to detect the incidence of pollination from plantation males using a set of diagnostic DNA markers that can distinguish wild seedlings from seedlings of hybrid origin.
The ability of hybrid seedlings to become established and grow can be an indicator of ecological competitiveness. To measure this, the number of hybrid and wild seedlings growing in artificially disturbed plots located adjacent to two hybrid plantations is compared with seed input monitored by seed traps placed next to the plots. Preliminary data indicate that proportion of established hybrid seedlings was significantly greater than the proportion of hybrid seeds arriving at each plot, and overwinter survival was significantly greater for hybrids than for wild seedlings. These results suggest that hybrid progeny were competitively superior to wild progeny. The experiments will be continued and expanded next year.
Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
ANIMAL RESEARCH NEWS
CATTLE CLONING ADVANCES WITH INTRODUCTION OF 'GENE'
A healthy six-month-old bull calf named Gene was introduced to the world in an August 7 press
release. The calf was produced through proprietary cloning technology developed by ABS Global,
Inc. of DeForest, Wisconsin. While excitement over the successful cloning of animals was fanned by
the popular press with news about a world famous sheep named Dolly, the fine points of reproducing
identical animals are still being worked out.
What makes Gene significant is not so much his cellular origin as the fact that there can be thousands of Genes. ABS Global has devised a method to divert primordial stem cells from their programmed path and instead become permanent embryonic stem cells. This means that each cell can be multiplied into an unlimited number of cells which can be stored frozen. Upon thawing, the nucleus from one of these cloned cells is substituted into a normal bovine egg cell which has had its own nucleus removed. After a week's growth in the test tube, the egg will have become an immature embryo that can be transferred to a recipient cow. About 280 days later, the cloned calf is born. Taking the remaining frozen cells through the same process will ultimately produce a herd of identical cattle.
The ultimate value in cloning animals depends first on having a superior or rare individual, one with proven exceptional performance or having a one-of-a-kind trait. Gene originated with cells taken from a 30-day-old fetus that was to some extent a genetic unknown. In contrast, the procedure used to clone Dolly began with an adult cell nucleus and essentially duplicated an adult animal whose characteristics were known. To make more 'Dollys' would require going through the entire procedure starting, in this case, with tissue from the mammary gland of a pregnant ewe.
ABS Global's proprietary cloning technology, which has been under development for more than ten years, thus opens the door to significantly increasing the capacity to produce genetically identical animals while substantially lowering the cost of doing so. Cloning technology could improve beef or dairy herd management since all cows will respond similarly to nutrition and the environment. Cows with particular traits, such as milk that makes the best mozzarella cheese, could be cloned to produce a 'niche market' herd. These and many other applications will improve the efficiency and profitability of dairy and beef operations.
Cloning has the potential to bring substantial economies of scale to the production of scarce or expensive pharmaceutical compounds in genetically engineered animals. Instead of a single million-dollar transgenic dairy cow producing a valuable recombinant protein in her milk, there can be a whole herd. ABS Global, Inc. has formed a separate business unit, Infigen, Inc., to commercialize applications of cloning technologies in the cattle breeding, pharmaceutical, nutraceutical and xenotransplantation fields.
Pat Traynor
Information Systems for Biotechnology
traynor@nbiap.biochem.vt.edu
INDUSTRY NEWS
STUDY SHOWS SIZABLE POTENTIAL IMPACT OF AGBIOTECH IN EUROPE
How does one calculate the value of agricultural biotechnology? As difficult as this may be, a group
out of the University of Sussex in the U.K. has made a go of it in their recent study, "Benchmarking
the Competitiveness of Biotechnology in Europe." Authors Burke and Thomas estimate that the
potential market for biotechnology-related products within the European Union will be nearly $280
billion by the year 2005. In addition, by their calculations, this equates to approximately three million
jobs. Given the magnitude of these predictions, some explanation is required.
A cornerstone of their estimates relies on their belief that there is tremendous growth potential in the agricultural and food sectors of biotechnology. They argue that less than 0.02% of the total European Gross Domestic Product (GDP) is made up by biopharmaceuticals, though this sector garners much of the attention. The agriculture and food sectors makes up a much larger portion of the GDP. In the Netherlands, for example, 20% of consumer spending and 18% of the work force is in these sectors. Burke and Thomas predict that 70% of biotechnology growth will come from agriculture and food sectors, rising from the current level of nearly $45 billion to the predicted level of nearly $280 billion by 2005.
The estimate of new jobs was generated by first determining what one job is worth in GDP dollars, and then dividing this value into the predicted value for biotechnology-related products in 2005. This yields an estimated increase in biotechnology-dependent jobs from 588,000 (calculated using 1995 data) to three million.
The authors base their predictions on a number of assumptions, including:
Burke and Thomas note that for their predictions to be realized, a number of changes must occur within the European regulatory framework. These include modifying regulations to level the playing field so that European farmers have the same access to technology as farmers in other countries. Also, there needs to be greater transparency in regulations, and finally, European farmers must be exposed to world prices. Without such changes, Burke and Thomas, and others, fear that Europe will be unable to take advantage of a sizable economic opportunity.
Reference
1. Burke, J. F. and Thomas, S. M. Agriculture is biotechnology's future in Europe. Nature Biotechnology, Volume 15, August 1997, pg. 695-696.
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.
Information Systems for Biotechnology, 120 Engel Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308, tel: 540-231-2620, fax: 540-231-2614, email: traynor@nbiap.biochem.vt.edu
For internet access to the News Report, textfiles, and databases use one of the following procedures.
1. Through WWW: http://www.isb.vt.edu/
2. To have the News Report automatically emailed, send an email message to news@nbiap.biochem.vt.edu and type subscribe newsreport [your-name] in the message section.
ISB News Report
120 Engel Hall
Virginia Tech
Blacksburg, VA 24061-0308