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[SANET-MG] not transgenic high lysine maize
http://www.i-sis.org.uk/highlysinemaize.phpISIS Press Release 23/11/05
Why Not Transgenic High Lysine Maize
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 lacks lysine
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 . 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 . Interestingly, piglets discriminated
between diets rich and poor in lysine and selected the lysine rich diets
Efforts to increase lysine in maize
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 .
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 .
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 . Growth inhibition and DNA damage are found in
mammalian cells treated with Cre recombinase . 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
 (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 . 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.
Terminator Recombinase Does Scramble Genomes
Dr. Mae-Wan Ho
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
. 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 . The conventionally bred high lysine
maize has also gone much further to accommodate the needs of indigenous
farmers for high lysine maize .
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 . 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
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
Salvador R. Maize 2005 http://maize.agron.iastate.edu/maizearticle.html
FAO Maize in human nutrition FAO Document Repository 1992
FAO Protein Sources for the Animal Feed Industry 2002
Clark HE, Bailey LB. and Brewer MF. Lysine and tryptophan in
cereal-based diets for adult human subjects. Am J Clin Nutr. 1977,
Kirchgessner M, Stangl G. and Roth F. Evidence for specific dietary
selection for lysine by the piglet J. Anim. Physiology and Anim.
Gibbon B and Larkins B. Molecular genetic approaches to developing
quality protein maize Trends in Genetics 2005, 21, 227-33.
Lucas,D. Petition for determination of nonregulated status for lysine
maize LY038 USDA/APHIS 2004
Coppoolse ER, de Vroomen MJ, Roelofs D, Smit J, van Gennip F, Hersmus
BJ, Nijkamp HJ and van Haaren MJ. Cre recombinase expression can result
in phenotypic aberrations in plants. Plant Mol Biol. 2003, ;51(2), 263-79.
Loonstra A, Vooijs M, Beverloo HB, Allak BA, van Drunen E, Kanaar R,
Berns A and Jonkers J. Growth inhibition and DNA damage induced by Cre
recombinase in mammalian cells. Proc Natl Acad Sci U S A. 2001, 31,
Ho MW. Terminator recombinase does scramble genomes. ISIS News 2001,
Schmidt EE, Taylor DS, Prigge JR, Barnett S and Capecchi MR.
Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse
spermatids. PNAS 2000, 97, 13702-13707.
Toride Y. Lysine and other amino acids for feed production and
contribution to protein utilization in animal feeding FAO Protein
Sources for the Animal Feed Industry 2002
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