UNAPPROVED CORN SLIPS THROUGH REGULATORY NET

On September 18, the Washington Post disclosed that Taco Bell brand taco shells, sold in the US, tested positive for StarLink corn, which is approved only for use in animal feed. Sold by Aventis CropScience, StarLink is engineered to contain the Cry9C protein obtained from Bacillus thuringiensis (Bt). By Friday, September 22, Kraft Foods voluntarily recalled its Taco Bell taco shells from stores. As a result of the incident, Kraft is recommending that crops approved for animal feed should not be allowed to enter the market unless they have also been approved for use in food, and is calling for a mandatory review of all new biotech crops.

There are no known health risks associated with the StarLink corn; however, because of questions about the allergenic potential of the Cry9C protein, the Environmental Protection Agency has not approved StarLink for use in foods meant for human consumption. Steve Taylor, Head of the Department of Food Science and Technology, University of Nebraska, is concerned that the detection of Cry9C in taco shells could be misunderstood by the public or food industry and offers the following information:

"StarLink was not approved for food use because the product did not pass all screens for allergenicity. The Bt protein in StarLink, Cry9C, does not resemble known allergens, so in fact it may not be an allergen. However, Cry9C was not immediately broken down in digestion tests. Other Bt products on the market contain a Cry1 protein, which is digested in a matter of seconds and has passed other screens for allergenicity. Furthermore, Cry1 proteins have been present in foods via Bt sprays used by farmers and home gardeners for many years.

"Was the public at risk because of this incident? I believe not. In order for people to become allergic to a protein they must be exposed to it multiple times over an extended period until they become sensitized. The protein must also be present as a relatively high percentage of total protein content. Most allergenic proteins are present at levels of one to 40 percent. Aventis indicates that the Cry9C protein is present in corn kernels at 0.3 percent, but the taco shells would contain far less due to the presence of other varieties of corn and the use of other ingredients. It is highly unlikely that Cry9C protein would be present in any corn products at a level of concern.

"It is important to understand that only a very small amount of StarLink corn was planted, about 300,000 acres among the nearly 80 million corn acres in the United States (0.3 of a percent). That small amount could conceivably be produced by only 100 large farms. Because of the feed-only restriction, nearly all would have been properly channeled to feed operations, but even if the production from one or two farms was improperly channeled, there would be only a few thousand acres to be co-mingled with other grain. This clearly would not produce protein levels of any health concern. In addition, a recent news release indicates that Aventis is going to buy back all of the StarLink corn to assure that any further amounts do not end up in the food chain."

COUNCIL FOR BIOTECHNOLOGY INFORMATION REQUEST FOR PROPOSALS

The purpose of this project is to establish an independent Web site that will allow scientists and consumers to more easily access data, facts, and information related to crop biotechnology from a variety of sources.

The Council for Biotechnology Information (CBI) will award a competitive grant to support or supplement the establishment and maintenance of an informational Web site that will serve as a comprehensive, evergreen repository of information on the safety of crops and foods derived using biotechnology. At least one full-time position will be required to collect information and keep the site updated. The grant recipient(s) will be expected to identify relevant and credible sources of information including peer-reviewed articles and information submitted according to regulatory data requirements. The institution(s) also will develop or work with experts to generate scientific summaries to help guide the viewer through the submission process required by regulatory agencies. A system will be established to post these items in a timely manner.

POTATO PACKS HIGH-PROTEIN PUNCH

The production of transgenic crops containing proteins with improved amino acid composition should be of benefit to humans as well as to monogastric animals (pig, poultry, etc.), who are unable to synthesize all the amino acids needed to sustain life. The potato is the most important non-cereal food crop in the world; however it contains limited amounts of the essential amino acids lysine, tryptophan, methionine, and cysteine. So far, genetic engineering experiments for quality improvement in potato have been focused on promoting pest and disease resistance, increasing yield, and managing biotic and abiotic stresses. Efforts to improve the nutritional content of potato have lagged. Improvements in the nutritional value of food crops such as potato are especially important for people subsisting on a vegetarian diet in which the main source of protein comes from seeds, grains, tubers, etc., which contain limiting amounts of essential amino acids.

A team of scientists from Jawaharlal Nehru University, New Delhi has reported improvement in the nutritive value of transgenic potato through the expression of a nonallergenic seed albumin gene (AmA1) from Amaranthus hypochondriacus. This gene, which was previously cloned and patented by the reporting team, encodes a seed-specific albumin. As a donor gene, the AmA1 gene has several advantages for genetic transformation experiments. First, this seed protein has a well-balanced amino acid composition, making it nutritionally superior to other proteins recommended by World Health Organization (WHO). Second, the purified protein has no known allergenic properties; and, finally, the protein is controlled by a single gene, which facilitates integration into other species.

For the plant transformation experiments, potato (Solanum tuberosum L. var A16) shoot cultures and Agrobacterium tumefaciens containing AmA1 expression plasmids were used. To achieve expression of the introduced gene in a tuber specific and constitutive manner, the team used granule-bound starch synthase (GBSS) and cauliflower mosaic virus (CaMV) 35S promoters, respectively. For each transformation, 40 independent transgenic plants were regenerated. Detection of the presence of the AmA1 gene in the transgenic plants by PCR amplification indicated the independent transformation status of the transgenics. Transgenic plants containing up to three copies of the transgene were transferred to the field and allowed to set tubers.

The transcripts were more plentiful in tubers than stem and leaf tissues in the transgenic lines, evidently indicating the greater activity of the GBSS promoter in tubers and stolons than in leaves. Also there was a five- to ten-fold increase in the transcript level in tubers of transgenic lines containing the GBSS promoter (pSB8G) compared to tubers from lines in which the constitutive 35S promoter (pSB8) was used. However, the expression of AmA1 among the transgenic lines was varied because of position effects.

The subcellular localization of AmA1 protein in both pSB8 and pSB8G tubers was studied to determine the basis for the stability of AmA1 protein in tuber cells of the transgenic plants. In wild type tubers, no signal for such localization was detected. However, AmA1 protein in transgenic plants was found to be present mainly in the cytoplasm and to a lesser extent in the vacuoles and was not accumulated into protein bodies.

Amino acid analysis was carried out in five pSB8G and six pSB8 highly expressing transgenic lines. When compared to the wild type A16 genotype, an increase in the essential amino acid content was reported in these selected transgenic lines. The pSB8 transgenic lines of the highly expressing tubers showed a significant 2.5- to 4-fold increase in lysine, methionine, cysteine, and tyrosine content.

Experiments were carried out in a restricted experimental plot in which four A16 wild type potato plants, 16 independent pSB8 transgenic lines, and eight independent pSB8G transgenic lines were grown. Data collected for two consecutive years showed consistent results. A 35 to 45% increase in total protein content was reported in transgenic tubers, which corresponded to an increase in most essential amino acids. A two-fold increase in tuber number and 3.0- to 3.5-fold increase in tuber yield was reported in both pSB8G and pSB8 lines.

To test for allergenicity, mice were injected with the purified AMA1 protein intranasally and intraperitoneally at three one-week intervals. This hypersensitivity test did not evoke any detectable IgE response. In addition, the team could not find any AmA1 sequence homology to allergic proteins recorded in Protein Data Bank. The team reported that a literature search did not reveal any allergenicity associated with amaranth grain or forage. Grain amaranth is used in many foods throughout the world and amaranth forage has been used for centuries as an important component of the human diet throughout the tropics. The authors presented these facts as evidence of the nonallergenic nature of amaranth.

The team was successful in using the seed albumin gene with a well-balanced amino acid composition as a donor protein to develop a nutritively improved transgenic potato. The study thus revealed the potential for biotechnological advancements to improve human nutrition. This knowledge will likely lead to the genetic modification of other crop plants with desired seed protein composition.

Source

BUILDING BETTER TREES THROUGH HORMONES

Plant hormones play critical roles in the growth, development, maturation, and senescence of plants. The ability to genetically regulate hormone levels provides researchers with a powerful tool for altering the outcomes of agricultural plant growth. Thomas Moritz and his team at Umeå Plant Science Centre in Sweden were able to increase wood biomass production in trees by enhancing gibberellin production¹.

Prior to the Mortiz's study, genetic engineering of trees was primarily limited to producing trees that flowered earlier, were pest and herbicide resistant, and produced wood with preservative properties. Little was done using gene technology to precisely manage hormone levels in trees. Hedden and Phillips, of the Long Ashton Research Station of Bristol in the United Kingdom, recently commented that enough is known about plant hormone biosynthesis for investigators to be able to manipulate them in commercial plants². They added that many of the hormone-encoding genes have been cloned and are ready for exploitation in crops.

The hormone gibberellin is of particular interest to researchers because of its broad range of effects on plant growth and development. Several gibberellin studies in transgenic Arabidopsis, barley, tobacco, and other research models support its utility in improving commercially important plants. Recent studies elucidating gibberellin regulation have resulted in an improved ability to selectively control gibberellin biosynthesis in transgenic plants³.

The gibberellins are comprised of a group of terpenoid hormones called the gibberellic acids (GA). These 19- to 20-carbon compounds are synthesized in plants from four isoprene units. Many of the GAs are the precursors to the biologically active forms; some are glycosylated for regulation or short-term storage and transport purposes. GAs are most noted for influencing stem elongation and certain aspects of maturation such as flower stalk bolting and flower induction. Gibberellins are also associated with the timing of seed germination.

Moritz's team focused on the role of gibberellin to regulate xylem deposition in trees¹. Gibberellin biosynthesis was stimulated by overexpressing GA 20-oxidase, a key enzyme responsible for the production of biologically active GAs from inactive 20-carbon precursor gibberellins. Initial studies on Arabidopsis showed that regulating GA 20-oxidase could successfully modify gibberellin levels. The result of this technology is comparable to the traditional practice of applying exogenous gibberellins to modify plant growth.

One to three copies of the AtGA20ox1 gene from Arabidopsis was inserted into the genome of hybrid aspen trees in tissue culture using a pPCV702.kana vector controlled by a CaMV 35S promotor. GA 20-oxidase overexpression was achieved in ten transformed hybrid aspen lines, and the GA content in leaves and internodes was determined.

Transgenic trees with the AtGA20ox1 gene showed higher levels of GAs in both leaves and internodes, demonstrating that hormone biosynthesis was enhanced in the plants. The bioactivity of gibberellin was confirmed by measuring increases in weight of the stem and internodal tissues. Leaf size was greater in the transgenic trees; however, there was no significant change in the number of nodes. The researchers achieved a 71% increase in xylem fiber number and an 8% increase in xylem fiber length in the transgenic trees. Moritz's team concluded that their technique was superior to using exogenous application of gibberellins. They showed an overall increase in plant growth as compared to exogenous application that results in increases in shoot growth at the expense of root growth.

This technique has potential for improving use of popular and underexploited commercial plants. Underexploited plants such as mesquite and kenaf could be altered using this technique to produce higher quality pulp than what is currently produced by the cultivated wild plants. Similar strategies with other hormones could enhance fruit production or tissue biomass for forage and industrial applications.

Sources

1. Eriksson ME, Israelsson M, Olsson O, and Mortiz T. 2000. Increased gibberellin biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length. Nature Biotechnology 18(7):784-788.

2. Hedden P and Phillips AL. 2000. Manipulation of hormone biosynthesis genes in transgenic plants. Current Opinions Biotechnology 11(2): 130-137.

3. Yamaguchi S and Kimiya U. 2000. Gibberellin biosynthesis: Its regulation by endogenous and environmental signals. Plant Cell Physiology 41(3):251-257.

THE TALE OF THE FIVE LITTLE PIGS AND XENA, THE PORCINE PRINCESS

In recent issues of Nature and Science, research groups from PPL Therapeutics and the National Institute of Animal Industry and Prima Meatpackers in Japan along with The Rockefeller University reported the cloning of pigs by nuclear transfer^1,2. Both of these groups used protocols that differed somewhat from that used for the production of Dolly, the cloned sheep. The PPL group utilized a double nuclear transfer procedure, whereas the Japanese/Rockefeller University research group used a piezo-actuated microinjection system. These reports are the first to add pigs to the growing list of mammals (mice, sheep, cattle, goats) that have been cloned by nuclear transfer.

TRANSGENIC ANIMALS NOW ELIGIBLE FOR CANADIAN PATENT PROTECTION

The Energizer^® rabbit is not the only rodent that keeps on going. Harvard's oncomouse has been running the maze of Canada's patenting process for fifteen years. At this time, it looks as if persistence will pay off. Twelve years after the US patent issued on the oncomouse, the Canadian patent is poised for issuance.

The journey began in 1985 when the President and Fellows of Harvard College filed a patent application with the Canadian Intellectual Property Office (CIPO), which claimed nonhuman transgenic mammals with germ cells and somatic cells that contained an activated oncogene. By 1993, the CIPO examiner had decided to allow claims to the process for producing the transgenic mammals, but had finally rejected claims to the mammals themselves. The CIPO took the position that transgenic animals are not patentable under the Canadian Patent Act, which is silent about the patentability of animals per se. Moreover, the CIPO did not consider animals to fit into the patentable categories of "manufacture" or "composition of matter," as Harvard had been arguing. The CIPO found support for this limiting interpretation of patentability in the Federal Court of Appeal's decision, Pioneer Hi-Bred v. Canada (Commissioner of Patents).

On August 4, 1995, after review by the CIPO Commissioner of Patents and an oral hearing before the Patent Appeal Board, the CIPO confirmed the rejection of transgenic mammal claims. Harvard appealed the decision in the Federal Court Trial Division. Here, the judge decided that there was no dispute that the oncomouse met the patentability requirements of novelty, usefulness, and nonobviousness. Nevertheless, the question was whether the transgenic animal is an "invention" as defined by the Patent Act. Dismissing the appeal, the judge explained that the inventors of the oncomouse could not impose sufficient control over their creation. That is, once the oncogene was introduced, everything else about the oncomouse was independent of human intervention. Accordingly, the transgenic mammal was not sufficiently uniformly reproducible to be considered as a "composition of matter" or an "article of manufacture." This was in April of 1998. Harvard appealed the decision.

Last August, the Federal Court of Appeal, in a 2-1 decision, sent the matter back to the CIPO with the direction to grant a patent on the transgenic animal claims. Writing for the court, Justice J.A. Rothstein explained that the oncomouse is eligible for patent protection because the animal is, literally, a composition of matter. The judge decided that nothing in the term "composition of matter" suggests that living things, particularly higher (nonhuman) life forms, are excluded from the definition. In adopting an expansive meaning of "composition of matter," Justice Rothstein acknowledged the persuasiveness of the US Supreme Court decision, Diamond v. Chakrabarty.

The judge also distinguished the oncomouse invention from the Pioneer Hi-Bred case, which concerned the crossbreeding of soybeans. Here, the significant difference is that the transgenic animal invention required "inventive ingenuity and intervention at the genetic level and the creation of a specific new life form."

With regard to the inventors' lack of control over all features of oncomice, the court found that control over characteristics such as eye color is irrelevant to the usefulness of the invention. In the end, Justice Rothstein explained that it is not up to the court to read limitations into the scope of patent legislation, and that Parliament could exclude higher life forms from patentability if it so desired.

Unless the Justice Department appeals the decision to the Supreme Court of Canada, the CIPO will need to deal with a backlog of about 250 patent applications on transgenic animals.

Sources