Look Mom! No Antibiotic Marker Genes!

Selectable marker genes are the sine qua non for the development of genetically modified crops, and all such commercially released GM crops have these marker genes. Marker genes render resistance in plant cells against antibiotics or herbicides and thus enable scientists to effortlessly select a rare transformed plant cell after co-introducing the desired gene along with a marker. Antibiotic or herbicide added to the plant culture media kills the normal plant cells while the few transformed cells survive, grow, and develop into whole plants.

Beyond the laboratory, these markers have no role and thus their presence in crops and food has provoked much public concern. The perceived risk of marker genes to environmental safety and health has led scientists to explore alternative systems such as the removal of marker genes using the Cre-lox system or transposable elements, and the use of cytokinin glucuronides with the GUS gene. A new selection strategy reported by Danish scientists involves a novel selectable marker system using a gene for an enzyme that metabolizes mannose-6-phosphate, a plant growth-inhibiting phosphorylated sugar, and thus the strategy may prove to be a better alternative to the use of antibiotic marker genes (1).

The most widely used selectable marker is the nptII gene that codes for neomycin phosphotransferase II, which confers resistance to antibiotic kanamycin or geneticin. Considerable research on the nptII has shown that it is safe for both the environment and the consumer (2). However, such information is not available for other marker genes including some herbicide resistance genes, which may carry a risk of potential gene flow to weedy relatives. There are also some concerns that antibiotic marker proteins could compromise the therapeutic efficiency of orally administered antibiotic.

The new selectable system relies on the use of phospho- mannose isomerase (PMI). PMI is fairly ubiquitous in nature; however, aside from leguminous plants, it is not found in most plants, especially cereals. Plant cells without this enzyme are unable to survive in a tissue culture medium containing mannose-6-phosphate as a sole carbon source. Incubation of plant cells in the presence of mannose-6-phosphate results in phosphate and ATP starvation thus depleting energy from critical functions such as cell division and elongation (3). The mannose-6-phosphate toxicity in plant cells has also recently been shown to cause apoptosis or programmed cell death through induction of an endonuclease responsible for DNA laddering (4).

The manA gene encoding PMI was cloned from E. coli by researchers at Danisco Biotechnology. Plant cells transformed with this gene can convert mannose-6-phosphate to fructose-6-phosphate, which is easily metabolized. Morten Joersbo and colleagues (1) observed that use of mannose selection in sugar beet resulted in a ten-fold increase in transformation frequency when compared to traditional kanamycin selection. According to researchers, such increased efficiency may occur because transformed cells are actively encouraged to grow rather than just allowed to survive.

In the traditional selection system using antibiotics or herbicides, the transgenic cells convert the selective agent to a detoxified compound that may still exert a negative influence on the plant cells. Further, the release of toxic metabolites by dying adjacent cells may also inhibit the growth of transformed cells. In contrast, mannose selection essentially provides a metabolic advantage to the transformed cells while the untransformed cells are starved and progressively lose their viability, according to Joersbo et al.

Novartis Agribusiness Biotechnology Research, Inc., which has licensed the PMI gene selection system, has found this marker to be very effective in the selection of wheat and maize transgenics with an astoundingly high frequency of transformation of 25% and 50%, respectively. Novartis scientists have found that PMI protein is very safe as is evident from many studies including mouse toxicity assays (3). The protein was readily digested in simulated mammalian gastric and intestinal fluids indicating a low allergenic potential. Sugar beet and maize plants containing the manA gene had identical biochemical profiles, yield, and nutritional composition when compared to control plants. The gene encoding this activity has been cloned from several bacteria and yeast species and also from humans. PMI has also been purified and studied from yeast, bacteria, pigs, and humans.

Supreme Court Backs States' Immunity

The 1992 Patent and Plant Variety Protection Remedy Clarification Act granted individuals the right to sue states for patent infringement. But, in a recent five to four decision, the US Supreme Court ruled that Congress lacked sufficient grounds to enact the legislation (2). This means that states have recovered an immunity to patent infringement actions in federal court.

In brief, here's what happened. Since 1987 College Savings has sold a certificate of deposit to help individuals save money for the cost of college education. Florida Prepaid Postsecondary Education Expenses Board, an entity created by the State of Florida, administers a similar investment program aimed at aiding individuals to fund the cost of Florida public colleges and universities.

In November 1994, College Savings brought an infringement action against Florida Prepaid in a US district court. College Savings claimed that in the course of administering its investment program Florida Prepaid infringed College Savings' patent. Florida Prepaid moved to dismiss the action arguing that the Patent Remedy Act did not provide a basis for dragging Florida Prepaid into court since the Act was an unconstitutional attempt by Congress to abolish state sovereign immunity. The district court denied Florida Prepaid's motion to dismiss the case, and Florida Prepaid appealed the decision to the Court of Appeals for the Federal Circuit. The Federal Circuit affirmed the lower court decision.

Florida Prepaid then appealed to the US Supreme Court where it found a sympathetic ear. Writing for a bare majority, Chief Justice William Rehnquist explained that the underlying conduct at issue is state patent infringement and a state's use of sovereign immunity to deny patent owners compensation for the invasion of their patent rights. The Court decided that, in enacting the Patent Remedy Act, Congress had failed to identify any pattern of patent infringement by states. The Court also noted that House Subcommittee testimony had acknowledged states are willing and able to respect patent rights. The Patent Remedy Act, therefore, cannot be viewed as remedial or preventive legislation aimed at securing protection for patent owners.

Government Responds to FOIA Targeting Research Data

Last October, Senator Richard Shelby (R-AL) inserted a provision into the Treasury and General Government Appropriations Act to order the Director of the Office of Management and Budget (OMB) to amend OMB Circular A-110. According to the amendment, federal awarding agencies would be required "to ensure that all data produced under an award will be made available to the public" through procedures established under the Freedom of Information Act (FOIA). The objective of the provision was to provide the public with access to the research and underlying data used by the federal government in developing policy and rules.

The Shelby amendment was not popular. As Representative George E. Brown, Jr. (D-CA) observed, "it is ironic, that a provision which some have described as a sunshine provision was tucked into a 4,000-page bill in the dead of night" (3). Concern in the scientific community focused on the implication that "anyone," not just the federal agencies themselves, could ask grantees for "all data," whatever that phrase means. The perceived threat was that scientists would have to turn over copies of their computer disks or lab notebooks in response to anyone who files a FOIA request to an agency that funded the research project (4). This would mean that raw data could become public before researchers had the chance to analyze and publish their data in a peer-reviewed journal.

In February, the OMB published a proposal to revise Circular A-110. It stated "for data relating to published research findings produced under an award used by the Federal Government in developing policy or rules, the Federal awarding agency shall, within a reasonable time, obtain the requested data so that they can be made available to the public" (5). That is, the OMB proposed to limit the Shelby amendment to data underlying published findings that the federal government used to develop policy or rules.

The OMB received over 9,000 remarks on the proposal with many of the commentators voicing concerns about the impact of the revision on the conduct of scientific research (6). In response to the comments, the OMB published a second proposal that reflects the OMB's view that the Shelby amendment does not require scientists to make research data publicly available while the research is still ongoing (6). Comments on the proposal with its new limiting definitions are due by September 10, 1999. The OMB intends to issue a final rule on or before September 30th.