Monsanto Corporation has created a transgenic maize line that produces high levels of the essential amino-acid lysine and is seeking non-regulated status for it in the United States. Professor Joe Cummins explains why it is a non-starter.
Maize is a domesticated grass of tropical Mexican origin, and is the third most planted crop after wheat and rice. The largest producers of maize are the United States, China and Brazil. Maize evolved with humans and depends on humans for its very existence because the seeds require humans for dissemination from the cobs. Maize is mainly produced for food, feed and fodder. The main kinds of maize include flint, which makes up about 14 percent of world production, and has a very hard seed coat suitable where storage and germination conditions are poor. Flour maize is the preferred form for human consumption directly, or in tortilla and dumpling, and accounts for 12 percent of commercial production. Dent maize, with a characteristic dent in the dry kernel, is used as livestock feed, starch, syrup, oil and alcohol, and accounts for 73 percent of world production. Sweet corn is tinned, frozen or served fresh to humans; it along with pop corn make up about one percent of world maize production [1]. Genetically modified (GM) traits have been incorporated into the different kinds of maize by crossing them with the original GM maize.
While maize is a major source of food and feed worldwide, it is not a suitable single source of nutrition. Maize alone does not provide the essential amino acid, lysine, in sufficient quantities for the nutritional needs of humans and farm animals. Traditional maize diets were accompanied by dry beans to compensate for the deficiency of lysine in maize. Currently, maize flour and seeds are supplemented with lysine in areas of the world where the supplement can be afforded. The Food and Agriculture Organization of the United Nations has reviewed the nutritional deficiency in conventional maize and established requirements for the amino acid [2, 3]. Daily requirements for lysine have been set at 400 to 900 mg for men and 300 to 700 mg for women but benefits were gained when lysine was increased to 1800 mg per day then more gradually up to 3600 mg [4]. Interestingly, piglets discriminated between diets rich and poor in lysine and selected the lysine rich diets [5].
Efforts to increase maize lysine using traditional breeding have been pursued for many years. In 1964 the opaque2 mutants were found to produce elevated levels of lysine, essentially by decreasing zein storage proteins, allowing lysine-rich proteins to accumulate in the endosperm. The original mutants were unsuitable for commercial production, but introducing a battery of modifier genes improved the field and storage qualities of the maize. Breeding for quantitative trait loci have also resulted in the production of high-lysine maize in countries where it can impact human nutrition [6].
The mutant high lysine strains selected by conventional breeding have therefore succeeded in producing proteins richer in lysine than the existing maize lines.
Nevertheless Monsanto Corporation has created a GM maize line that employs a synthetic approximation of a bacterial gene to increase the level of lysine in the amino acid pools in the grain, and Monsanto has lodged a petition for non-regulated status for the GM maize in the United States. The maize line LY038 was developed usingrecombinant DNA techniques to integrate the cordapA sequence into the maize genome. The cordapA sequence (Monsanto’s designation for the GM insert) contains the coding sequence of DHDPS, the lysine-insensitive dihydrodipicolinate synthase enzyme derived from Corynebacterium glutamicum, placed under the control of the maize Glb1(Globulin 1) promoter to direct enzyme expression predominantly in the embryo, to increase the level of lysine in grain for animal feed applications. The DHPDS sequence driven by the Glb1 promoter was preceded by two synthetic linker sequences, an intron from rice actin gene and a chloroplast targeting sequence from maize DHPDS gene. In the primary construct was included next to genes for the high lysine trait a loxP (recombination site recognized by Cre recombinase) followed by a CaMV promoter driving a neomycin antibiotic resistance gene along with bleomycin resistance gene with a nos transcription terminator from Agrobacterium followed by another lox site. The lox sites flank the antibiotic resistance genes, to be cut out by the Cre recombinase added later by crossing maize lines. Finally, an ampicillin resistance gene with a bacterial promoter was included [7].
The basic idea of the construction was to introduce a bacterial enzyme that had a reduced feedback inhibition on lysine synthesis, allowing lysine to accumulate in the cellular amino acid pool.
The purpose of the lox recombination sites was to provide a means of removing the neomycin antibiotic resistance gene after it use in selection was no longer required (It is not clear why the ampicillin resistance gene was allowed to remain in the final strain).
Plants expressing the gene for Cre recombinase, however, are prone to abnormalities [8]. Growth inhibition and DNA damage are found in mammalian cells treated with Cre recombinase [9]. This Cre/lox system was used in many sterile seed (terminator) technologies in the early days, which force farmers to buy seed every year. It was also used to control breeding in animals. In one experiment with transgenic mice, it scrambled the genome so badly that the mice became completely sterile [10] (see Box).
A maize strain bearing the Cre recombinase gene was crossed with the high lysine transgenic maize line to remove the neomycin cassette. When it was established that the line lacked the neomycin cassette, the high lysine maize-Cre recombinase hybrid line was selfed, and by the F3 generation, plants lacking the Cre-recominase gene were selected and used to establish the final high lysine maize line [7]. There were no studies on the genetic damage and chromosome scrambling that undoubtedly took place during the time in which the Cre-recombinase was associated with the high lysine strain.
We predicted some time ago that the recombinase enzyme used in terminator technologies would scramble genomes (see "Terminator in new guises" ISIS News #3, 1999). This was demonstrated in transgenic mice engineered, which we reported in ISIS News #7/8, 2001).
The recombinase Cre is part of the ‘site-specific recombination’ Cre/lox system originally isolated from the bacteriophage (bacterial virus) P1. Cre catalyses recombination between two lox sites, splicing out any stretch of DNA in between. The lox site is a 34 basepair element consisting of 13 basepair inverted repeat separated by a core of 8 basepairs. In order to work, the 8 basepair core of the two lox sites have to be in the same orientation.
The system was not only used in plants, but was also extensively exploited in transgenic mice. Studies in the test-tube have shown that Cre recombinase can catalyse recombination between DNA sequences found naturally in yeast and mammalian genomes. These ‘illegitimate sites’ often bear little sequence similarity to the lox element.
Researchers in the United States showed that high levels of Cre expression in the sperm cells of heterozygous transgenic mice led to 100 percent sterility in the males, despite the absence of any lox sites [11]. Heterozygous mice carry only one copy of the Cre recombinase gene.
The sterility is caused directly by the recombinase enzyme scrambling the genome, essentially by breaking and rejoining DNA at inappropriate sites on the same or different chromosomes. The researchers pinpointed the genome-scrambling event to the time at which the two ‘daughter’ spermatids (precursors of sperms) and their paired chromosomes have just separated from each other; but are still joined by a ‘cytoplasmic bridge’. This was enough to allow the enzyme to pass from the spermatid containing the recombinase gene to the other lacking it, thereby to scramble up the chromosomes of both the transgenic and nontransgenic spermatid. The result was 100 percent sterility. Embryos fertilized by these sperms arrested predominantly at the 2-cell stage, and did not go beyond the four cell stage.
The researchers warn: “These results indicate that Cre can catalyze illegitimate recombination having overt pathological consequences in animals.” A similar recombination system is found in animals containing the RAG recombinases. There, illegitimate recombinations in somatic cells are linked to human leukemias.
Is transgenic high-lysine maize more economically advantageous compared to amino-acid supplement, or high lysine strains produced by conventional breeding? An analysis reported in a FAO workshop indicated that lysine supplement was far more economical source of lysine than was transgenic high lysine maize [12]. The conventionally bred high lysine maize has also gone much further to accommodate the needs of indigenous farmers for high lysine maize [6].
A full sequence of the transgenic DHPDS protein has not been presented, nor was there a search for allergenic epitopes in the protein structure. There has been no feeding experiment with the transgenic DHPDS protein, or for that matter, with the transgenic high lysine maize [7]. It is not known whether the lysine stored in cellular pools is stable, or that its availability is equivalent to the stored lysine-rich proteins during processing for food or feed.
Furthermore, genome-wide analyses of the transgenic high-lysine maize should be performed to detect genetic and other genome damages that may compromise safety or agronomic performance in the field.
The petition for non-regulated status should not be considered at least until these matters are cleared up. Public Comment on the Monsanto Proposal for High Lysine Maize can be made until 28 November 2005 at URL: http://www.regulations.gov/fdmspublic-bld61/component/main
This article has been submitted to oppose the petition for non-regulated status of Monsanto’s transgenic high lysine maize on behalf of the Independent Science Panel. Please register your objection and refer to this article.
Article first published 23/11/05
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