Open access to science eprints TheSparc provides both scientists and the general public free open access to scientific papers that are important for the survival of people and planet
ISIS Report - March 19 2001
Terminator insects give wings to genome invaders
The United States Department of Agriculture has approved field
release of GM pink bollworm this summer, which are made with a mobile genetic
element that can jump many species. This is tantamount to giving wings to the
most aggressive genome invaders. Dr. Mae-Wan Ho exposes evidence of
instability in these GM insects, and warns of rampant horizontal gene transfer
and recombination, should such GM insects become released.
Geneticists have created a particularly hazardous class of gene transfer
vectors for engineering insects. These are transposons, or mobile genetic
elements, which, as the name implies, are genetic units that can move from one
site to another in the same genome or move between genomes belonging to
unrelated species. Transposons are related to viruses and proviral sequences
that, like transposons, are found in the genomes of all species.
A transposon consists of several genes flanked by terminal repeat
sequences. One of the genes will code for the enzyme transposase, which is
necessary for moving the element. However, elements that have lost the
transposase gene can nevertheless get help from the enzyme coded in other
transposons. Transposons come in groups, or superfamilies, many of which have
members distributed widely across species belonging to different phyla of both
animals and plants. These promiscuous transposons have found
special favour with genetic engineers, whose goal is to create
universal systems for transferring genes into any and every species
on earth. Almost none of the geneticists has considered the hazards
A group in Boston created a vector from mariner, a superfamily of
transposons found across genomes of diverse species from insects to plants and
vertebrates, including human beings. One element belonging to this
superfamily, Hirmar1, isolated from the horn fly, was used to make
minitransposons consisting of the short inverted terminal repeats,
between which any gene expression cassette(s) can be inserted. The researchers
constructed a minitransposon with a kanamycin antibiotic resistance marker gene
driven by a bacterial promoter. This minitransposon was found to jump
easily into the E. coli and the Mycobacterium chromosome. It is
known to recognize the dinucleotide TA. The probability of this dinucleotide
occurring in any stretch of DNA is 0.252 or 6.25%. Within the 500
base pairs of the bacterial chromosome analysed, 21 of the 23 possible TA
dinucleotide insertion sites were occupied (1).
The experiment shows that the transposon can be stripped down to the
bare minimum of the flanking repeats, and it can still jump into genomes. The
reason, as mentioned earlier, is that the transposase function can be supplied
by a helper transposon. Such helper transposons are ubiquitous. So,
it would seem obvious that integrated transposon vectors may easily jump out
again, to another site in the same genome, or to the genome of unrelated
species. There are already signs of that in the transposon, piggyBac,
used in the GM bollworms to be released by the USDA this summer.
The piggyBac transposon was discovered in cell cultures of the
moth Trichopulsia, the cabbage looper, where it caused high rates of
mutations in the baculovirus infecting the cells by jumping into its genes (2).
The piggyBac is 2.5kb long with 13 bp inverted terminal repeats. It has
a specificity for sites with the base sequence TTAA. (The probability of this
sequence occurring is 0.254 or 0.4%.) This transposon was later
found to be active in a wide range of species, including the fruitfly
Drosophila, the mosquito transmitting yellow fever, Aedes
aegypti, the medfly, Ceratitis capitata, and the original host, the
cabbage looper (3). The piggyBac vector gave high frequencies of
transpositions, 37 times higher than mariner and nearly four times
higher than Hirmar.
In another experiment, the integrative piggyBac vector, with its
transposase gene disabled and carrying the green fluorescent protein gene
cassette, was used to transform the silkworm, Bombyx mori L (4).
Transposase function was provided by a helper-plasmid containing a
piggyBac transposon also disabled by having one of its terminal repeats
removed. The integrative vector and helper plasmids were both injected into
silkworm embryos. The adult fertile moths (G0) resulting from the injected
embryos were mated in single pairs among themselves or backcrossed to the
unmodified parent, and the resultant broods (G1) were analysed.
A total of 2498 embryos from two strains of silk worms were injected,
1164 (46.6%) of the embryos hatched resulting in 654 (26.7%) fertile adults,
single-pair matings among which 12 broods (0.5%) expressing green fluorescent
protein were found.
The genomic DNA of the broods were analysed with Southern blot (a
technique that gives information on the inserts). Here is how the authors
reported their results.
"Southern blot analyses of the DNA of transformed G1 insects showed that
one to three different inserts were present in a single animal and that larvae
from the same progeny [ie, brood] had different insertions. These insertions
were inherited independently at the G2 generation
"The presence of multiple independent inserts in many G1 larvae
indicates that a single gamete from the G0 parent can harbor several insertions
and that different gametes can have different insertions. Eighteen insertions
were observed in 12 G1 individuals issued from three transformed parents. It is
likely that this result underestimates the total number of insertion events
that occurred in the G0 moths." (p.82)
What was the explanation for such a large number of different inserts?
There were two possible explanations.
"Either the integration events [of the piggyBac vector] in the
germ line occurred late during development [of the injected embryo]", so that
the same adult carries a population of germ cells each with different
insertions, "or successive rounds of transposition took place after an initial
insertion event". The latter hypothesis, considered more likely, "would explain
why despite the low frequency of insertion in the parental population
[0.5%] the number of inserts is high in the transformed insects ..
A similar situation was also observed in transgenic C. capitata, and it
was also attributed to secondary mobilizations of an initial single insert."
In other words, there is evidence that the inserts had moved between the
G0 and the G1 generations, and possibly, again between the G1 and G2
generations. The "stable germ line transformation" claimed (p.83) is based on a
dangerous instability of the insert, which is prone to secondary mobilization.
These artificial transposons are already aggressive genome invaders, and
putting them into insects is to give them wings, as well as sharp mouthparts
for efficient delivery to all plants and animals and their viruses. The
predictable result is rampant horizontal gene transfer and recombination across
species barriers. The unpredictable unknown is what kinds of new deadly viruses
might be generated (5), and how many new cases of insertion mutagenesis and
carcinogenesis they may bring (6). It is the height of folly and
irresponsibility to release such GM insects, let alone GM insects carrying
female-killing genes (7).
Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, and Mekalanos
JJ. In vivo transpostion of mariner-based elements in enteric bacteria and
mycobacteria. Proc. Natl. Acad.Sci USA 1999: 96: 164-1650. See
also "Can such rampant gene shuffling be
safe?" Mae-Wan Ho and Angela Ryan, ISIS News 4, March 2000