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
The mutant high lysine strains selected by conventional breeding have therefore
succeeded in producing proteins richer in lysine than the existing maize lines.
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 DHPDSsequence
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 Agrobacteriumfollowed 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).
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).
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
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
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
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.
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.
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,