ISIS Report 19/08/00
Horizontal Gene Transfer The Hidden Hazards of
Dr. Mae-Wan Ho
A version of this paper will appear on the website of
SCOPE - a NSF-funded research project involving Science Journal
and groups at the University of California at Berkeley and the University
of Washington in Seattle.
Genetic engineering involves designing artificial constructs to cross
species barriers and to invade genomes. In other words, it enhances
horizontal gene transfer the direct transfer of genetic material to
unrelated species. The artificial constructs or transgenic DNA typically
contain genetic material from bacteria, viruses and other genetic
parasites that cause diseases as well as antibiotic resistance genes that
make infectious diseases untreatable. Horizontal transfer of transgenic
DNA has the potential, among other things, to create new viruses and
bacteria that cause diseases and spread drug and antibiotic resistance
genes among pathogens. There is an urgent need to establish effective
regulatory oversight to prevent the escape and release of these dangerous
constructs into the environment, and to consider whether some of the most
dangerous experiments should be allowed to continue at all.
Key words: antibiotic resistance genes, dormant viruses, CaMV promoter,
cancer, naked DNA, transgenic DNA,
Transgenic pollen and baby bees
Prof. Hans-Hinrich Kaatz from the University of Jena, is reported to
have new evidence, as yet unpublished, that genes engineered into
transgenic plants have transferred via pollen to bacteria and yeasts
living in the gut of bee larvae(1).
If Prof. Kaatz claim can be substantiated, it indicates that the
new genes and gene-constructs introduced into transgenic crops and other
transgenic organisms can spread, not just by ordinary cross-pollination or
cross-breeding to closely related species, but by the genes and
gene-constructs invading the genomes (the totality of the organisms
own genetic material) of completely unrelated species, including the
microorganisms living in the gut of animals eating transgenic material.
This finding is not unexpected. Some scientists have been drawing
attention to this possibility recently(2), but the warnings actually date
back to the mid-1970s when genetic engineering began. Hundreds of
scientists around the world are now demanding a moratorium on all
environmental releases of transgenic organisms on grounds of safety(3),
and horizontal gene transfer is one of the major considerations.
Some of us have argued that the hazards of horizontal gene
transfer to unrelated species are inherent to genetic engineering(4).
The genes and gene-constructs created in genetic engineering have
never existed in billions of years of evolution. They consist of genetic
material originating from bacteria, viruses and other genetic parasites
that cause diseases and spread drug and antibiotic resistance genes. They
are designed to cross all species barriers and to invade genomes. The
spread of such genes and gene-constructs have the potential to make
infectious diseases untreatable and to create new viruses and bacteria
that cause diseases.
Horizontal gene transfer may spread transgenes to the entire biosphere
Horizontal gene transfer is the transfer of genetic material between
cells or genomes belonging to unrelated species, by processes other than
usual reproduction. In the usual process of reproduction, genes are
transferred vertically from parent to offspring; and such a
process can occur only within a species or between closely related
Bacteria have been known to exchange genes across species barriers in
nature. There are three ways in which this is accomplished. In conjugation,
genetic material is passed between cells in contact; in transduction,
genetic material is carried from one cell to another by infectious
viruses; and in transformation, the genetic material is taken up
directly by the cell from its environment. For horizontal gene transfer to
be successful, the foreign genetic material must become integrated into
the cells genome, or become stably maintained in the recipient cell
in some other form. In most cases, foreign genetic material that enters a
cell by accident, especially if it is from another species, will be broken
down before it can incorporate into the genome. Under certain ecological
conditions which are still poorly understood, foreign genetic material
escapes being broken down and become incorporated in the genome. For
example, heat shock and pollutants such as heavy metals can favor
horizontal gene transfer; and the presence of antibiotics can increase the
frequency of horizontal gene transfer 10 to 10 000 fold(5).
While horizontal gene transfer is well-known among bacteria, it is only
within the past 10 years that its occurrence has become recognized among
higher plants and animals(6). The scope for horizontal gene transfer is
essentially the entire biosphere, with bacteria and viruses serving both
as intermediaries for gene trafficking and as reservoirs for gene
multiplication and recombination (the process of making new combinations
of genetic material (7)).
There are many potential routes for horizontal gene transfer to plants
and animals. Transduction is expected to be a main route as there are many
viruses which infect plants and animals. Recent research in gene therapy
indicates that transformation is potentially very important for cells of
mammals including human beings. A great variety of naked
genetic material are readily taken up by all kinds of cells, simply as the
result of being applied in solution to the eye, or rubbed into the skin,
injected, inhaled or swallowed. In many cases, the foreign gene constructs
become incorporated into the genome(8).
Direct transformation may not be as important for plant cells, which
generally have a protective cell wall. But soil bacteria belonging to the
genus Agrobacterium are able to transfer the T (tumour)
segment of its Tumour-inducing (Ti) plasmid (see below) into plant
cells in a process resembling conjugation. This T-DNA is widely
exploited as a gene transfer vehicle in plant genetic engineering (see
below). Foreign genetic material can also be introduced into plant and
animal cells by insects and arthropods with sharp mouthparts. In addition,
bacterial pathogens which enter plant and animal cells may take up foreign
genetic material and carry it into the cells, thus serving vectors for
horizontal gene transfer(9). There are almost no barriers preventing the
entry of foreign genetic material into the cells of probably any species
on earth. The most important barriers to horizontal gene transfer operate
after the foreign genetic material has entered the cell(10).
Most foreign genetic material, such as those present in ordinary food,
will be broken down to generate energy and building-blocks for growth and
repair. There are many enzymes which break down foreign genetic material;
and in the event that the foreign genetic material is incorporated into
the genome, chemical modification can still put it out of action and
However, viruses and other genetic parasites such as plasmids and
transposons, have special genetic signals and probably overall structure
to escape being broken down. A virus consists of genetic material
generally wrapped in a protein coat. It sheds its overcoat on entering a
cell and can either hi-jack the cell to make many more copies of itself,
or it can jump directly into the cells genome. Plasmids are pieces
of free, usually circular, genetic material that can be
indefinitely maintained in the cell separately from the cells
genome. Transposons, or jumping genes, are blocks of genetic
material which have the ability to jump in and out of genomes, with or
without multiplying themselves in the process. They can also land in
plasmids and be propagated there. Genes hitch-hiking in genetic parasites,
ie, viruses, plasmids and transposons, therefore, have a greater
probability of being successfully transferred into cells and genomes.
Genetic parasites are vectors for horizontal gene transfer.
Natural genetic parasites are limited by species barriers, so for
example, pig viruses will infect pigs, but not human beings, and
cauliflower viruses will not attack tomatoes. It is the protein coat of
the virus that determines host specificity, which is why naked viral
genomes (the genetic material stripped of the coat) have generally been
found to have a wider host range than the intact virus(11). Similarly, the
signals for propagating different plasmids and transposons are usually
specific to a limited range of host species, although there are
As more and more genomes have been sequenced, it is becoming apparent
that gene trafficking or horizontal gene transfer has played an important
role in the evolution of all species(12). However, it is also clear that
horizontal gene trafficking is regulated by internal constraints in the
organisms in response to ecological conditions(13).
Genetic engineering is unregulated horizontal gene transfer
Genetic engineering is a collection of laboratory techniques used to
isolate and combine the genetic material of any species, and then to
multiply the constructs in convenient cultures of bacteria and viruses in
the laboratory. Most of all, the techniques allow genetic material to be
transferred between species that would never interbreed in nature. That is
how human genes can be transferred into pig, sheep, fish and bacteria; and
spider silk genes end up in goats. Completely new, exotic genes are also
being introduced into food and other crops.
In order to overcome natural species barriers limiting gene transfer and
maintenance, genetic engineers have made a huge variety of artificial
vectors (carriers of genes) by combining parts of the most infectious
natural vectors viruses, plasmids and transposons - from different
sources. These artificial vectors generally have their disease-causing
functions removed or disabled, but are designed to cross wide species
barriers, so the same vector may now transfer, say, human genes spliced
into the vector, to the genomes of all other mammals, or of plants.
Artificial vectors greatly enhance horizontal gene transfer (see Box
Artificial vectors enhance horizontal gene transfer
- They are derived from natural genetic parasites that mediate
horizontal gene transfer most effectively.
- Their highly chimaeric nature means that they have sequence
homologies (similarities) to DNA from viral pathogens, plasmids and
transposons of multiple species across Kingdoms. This will
facilitate widespread horizontal gene transfer and recombination.
- They routinely contain antibiotic resistance marker genes which
enhance their successful horizontal transfer in the presence of
antibiotics, either intentionally applied, or present as xenobiotic
in the environment. Antibiotics are known to enhance horizontal gene
transfer between 10 to 10 000 fold.
- They often have origins of replication and transfer
sequences, signals that facilitate horizontal gene transfer
and maintenance in cells to which they are transferred.
- Chimaeric vectors are well-known to be structurally unstable, ie,
they have a tendency to break and join up incorrectly or with other
DNA, and this will increase the propensity for horizontal gene
transfer and recombination.
- They are designed to invade genomes, to overcome mechanisms that
breakdown or disable foreign DNA and hence will increase the
probability of horizontal transfer.
Although different classes of vectors are distinguishable on the basis
of the main-frame genetic material, practically every one of them is
chimaeric, being composed of genetic material originating from the genetic
parasites of many different species of bacteria, animals and plants.
Important chimaeric shuttle vectors enable genes to be
multiplied in the bacterium E. coli and transferred into species
in every other Kingdom of plants and animals. Simply by creating such a
vast variety of promiscuous gene transfer vectors, genetic engineering
biotechnology has effectively opened up highways for horizontal gene
transfer and recombination, where previously the process was tightly
regulated, with restricted access through narrow, tortuous footpaths.
These gene transfer highways connect species in every Domain and Kingdom
with the microbial populations via the universal mixing vessel used in
genetic engineering, E. coli. What makes it worse is that there is
currently still no legislation in any country to prevent the escape and
release of most artificial vectors and other artificial constructs into
the environment (15).
What are the hazards of horizontal gene transfer?
Most artificial vectors are either derived from viruses or have viral
genes in them, and are designed to cross species barriers and invade
genomes. They have the potential to recombine with the genetic material of
other viruses to generate new infectious viruses that cross species
barriers. Such viruses have been appearing at alarming frequencies. The
antibiotic resistance genes carried by artificial vectors can also spread
to bacterial pathogens. Has the growth of commercial-scale genetic
engineering biotechnology contributed to the resurgence of drug and
antibiotic infectious diseases within the past 25 years (16)?
There is already overwhelming evidence that horizontal gene transfer and
recombination have been responsible for creating new viral and bacterial
pathogens and for spreading drug and antibiotic resistance among the
pathogens. One way that new viral pathogens may be created is through
recombination with dormant, inactive or inactivated viral genetic material
that are in all genomes, plants and animals without exception.
Recombination between external and resident, dormant viruses have been
implicated in many animal cancers (17).
As stated earlier, the cells of all species including our own can take
up foreign genetic material. Artificial constructs designed to invade
genomes may well invade our own. These insertions may lead to
inappropriate inactivation or activation of genes (insertion mutagenesis),
some of which may lead to cancer (insertion carcinogenesis)(18). The
hazards of horizontal gene transfer are summarized in Box 2.
Potential hazards of horizontal gene transfer from genetic
- Generation of new cross-species viruses that cause disease
- Generation of new bacteria that cause diseases
- Spreading drug and antibiotic resistance genes among the viral
and bacterial pathogens, making infections untreatable
- Random insertion into genomes of cells resulting in harmful
effects including cancer
- Reactivation of dormant viruses, present in all cells and
genomes, which may cause diseases
- Spreading new genes and gene constructs that have never existed
- Multiplication of ecological impacts due to all of the above.
Transgenic DNA may be more likely to transfer horizontally than
Both the artificial vectors used in genetic engineering and the genes
transferred to make transgenic organisms are predominantly from viruses
and bacteria associated with diseases, and these are being brought
together in combinations that have never existed in billions of years of
Genes are never transferred alone. They are transferred in
unit-constructs, known as an expression cassettes. Each gene
has to be accompanied by a special piece of genetic material, the promoter,
which signals the cell to turn the gene on, ie, to transcribe the DNA gene
sequence into RNA. At the end of the gene there has to be another signal,
a terminator, to end the transcription and to mark the RNA, so it
can be further processed and translated into protein. The simplest
expression cassette looks like this:
Typically, each bit of the construct: promoter, gene and terminator, is
from a different source. The gene itself may also be a composite of bits
from different sources. Several expression cassettes are usually linked in
series, or stacked in the final construct. At least one of the
expression cassettes will be that of an antibiotic resistance marker gene
to enable cells that have taken up the foreign construct to be selected
with antibiotics. The antibiotic resistance gene cassette will often
remain in the transgenic organism.
The most commonly used promoters are from viruses associated with
serious diseases. The reason is that such viral promoters give continuous
over-expression of genes placed under their control. The same basic
construct is used in all applications of genetic engineering, whether in
agriculture or in medicine, and the same hazards are involved. There are
reasons to believe that transgenic DNA is much more likely to spread
horizontal than the organisms own DNA (see Box 3) (19).
Reasons to suspect that transgenic DNA may be more likely to
spread horizontally than non-transgenic DNA
- Artificial constructs and vectors are designed to be invasive to
foreign genomes and overcome species barriers.
- All artificial gene-constructs are structurally unstable (20),
and hence prone to recombine and transfer horizontally.
- The mechanisms enabling foreign genes to insert into the genome
also enable them to jump out again, to re-insert at another site, or
to another genome.
- The integration sites of most commonly used artificial vectors
- genes are recombination hotspots, and so have an
increased propensity to transfer horizontally.
- Viral promoters, such as that from the cauliflower mosaic virus,
widely used to make transgenes over-express, contain recombination
hotspots (21), and will therefore further enhance horizontal gene
- The metabolic stress on the host organism due to the continuous
over expression of transgenes may also contribute to the instability
of the insert (22).
- The foreign gene-constructs and the vectors into which they are
spliced, are typically mosaics of DNA sequences from numerous
species and their genetic parasites; that means they will have
sequence homologies with the genetic material of many species and
their genetic parasites, thus facilitating wide-ranging horizontal
gene transfer and recombination.
Additional hazards from viral promoters
We have recently drawn attention to additional hazards associated with
the promoter of the cauliflower mosaic virus (CaMV) most widely used in
agriculture (23). It is in practically all transgenic plants already
commercialized or undergoing field trials, as well as a high proportion of
transgenic plants under development, including the much acclaimed golden
CaMV is closely related to human hepatitis B virus, and less so, to
retroviruses such as the AIDS virus (25). Although the intact virus itself
is infectious only for cruciferae plants, its promoter is promiscuous in
function, and is active in all higher plants, in algae, yeast, and E.
coli (26), as well as frog and human cell systems (27). Like all
promoters of viruses and of cellular genes, it has a modular structure,
with parts common to, and interchangeable with promoters of other plant
and animal viruses. It has a recombination hotspot, flanked by multiple
motifs involved in recombination, similar to other recombination hotspots
including the borders of the Agrobacterium T DNA vector most
frequently used in making transgenic plants. The suspected mechanism of
recombination requires little or no DNA sequence homologies. Finally,
viral genes incorporated into transgenic plants have been found to
recombine with infecting viruses to generate new viruses (28). In some
cases, the recombinant viruses are more infectious than the original.
Proviral sequences generally inactive copies of viral genomes -
are present in all plant and animal genomes, and as all viral promoters
are modular, and have at least one module the TATA box - in common,
if not more. It is not inconceivable that the CaMV 35S promoter in
transgenic constructs can reactivate dormant viruses or generate new
viruses by recombination. The CaMV 35S promoter has been joined
artificially to copies of a wide range of viral genomes, and infectious
viruses produced in the laboratory (29). There is also evidence that
proviral sequence in the genome can be reactivated (30).
These considerations are especially relevant in the light of recent
findings that certain transgenic potatoes - containing the CaMV 35S
promoter and transformed with Agrobacterium T-DNA - may be unsafe
for young rats, and that a significant part of the effects may be due to "the
construct or the genetic transformation (or both) (31)" The authors
also report an increase in lymphocytes in the intestinal wall, which is a
non-specific sign of viral infection (32).
Evidence for horizontal transfer of transgenic DNA
It is often argued that transgenic DNA, once incorporated into the
transgenic organism, will be just as stable as the organisms own
DNA. But there is both direct and indirect evidence against this
supposition. Transgenic DNA is more likely to spread, and has been found
to spread by horizontal gene transfer.
Transgenic lines are notoriously unstable and often do not breed true
(33). There is a paucity of molecular data documenting the structural
stability of the transgenic DNA, both in terms of its site of insertion in
the genome and its arrangement of genes, in successive generations.
Instead, transgenes may be silenced in subsequent generations or lost
A herbicide-tolerance gene, introduced into Arabidopsis by means
of a vector, was found to be up to 30 times more likely to escape and
spread than the same gene obtained by mutagenesis (35). One way this may
happen is by secondary horizontal gene transfer via insects visiting the
plants for pollen and nectar (36). The reported finding that pollen can
transfer transgenic DNA to bacteria in the gut of bee larvae is relevant
Secondary horizontal transfer of transgenes and antibiotic resistant
marker genes from genetically engineered crop-plants into soil bacteria
and fungi have been documented in the laboratory. Transfer to fungi was
achieved simply by co-cultivation (37), while transfer to bacteria has
been achieved by both re-isolated transgenic DNA or total transgenic plant
DNA (38). Successful transfers of a kanamycin resistance marker gene to
the soil bacterium Acinetobacter were obtained using total DNA
extracted from homogenized plant leaf from a range of transgenic plants:
Solanum tuberosum (potato), Nicotiana tabacum (tobacco),
Beta vulgaris (sugar beet), Brassica napus (oil-seed rape)
and Lycopersicon esculentum (tomato) (39). It is estimated that
about 2500 copies of the kanamycin resistance genes (from the same number
of plant cells) is sufficient to successfully transform one bacterium,
despite the fact that there is six million-fold excess of plant DNA
present. A single plant with say, 2.5 trillion cells, would be
sufficient to transform one billion bacteria.
Despite the misleading title in one of the publications,(40) a high gene
transfer frequency of 5.8 x 10-2 per recipient bacterium was demonstrated
under optimum conditions. But the authors then proceeded to calculate an
extremely low gene transfer frequency of 2.0 x 10-17 under extrapolated "natural
conditions", assuming that different factors acted independently.
The natural conditions, however, are largely unknown and unpredictable,
and even by the authors own admission, synergistic effects cannot be
ruled out. Free transgenic DNA is bound to be readily available in the
rhizosphere around the plant roots, which is also an environmental
hotspot for gene transfer (41). Other workers have found evidence of
horizontal transfer of kanamycin resistance from transgenic DNA to Acinetobactor,
and positive results were obtained using just 100ml
of plant-leaf homogenate (42).
Defenders of the biotech industry still insist that just because
horizontal gene transfer occurs in the laboratory does not mean it can
occur in nature. However, there is already evidence suggesting it can
occur in nature. First of all, genetic material released from dead and
live cells, is now found to persist in all environments; and not rapidly
broken down as previously supposed. It sticks to clay, sand and humic acid
particles and retains the ability to infect (transform) a range of
micro-organisms in the soil (43). The transformation of bacteria in the
soil by DNA adsorbed to clay sand and humic acid has been confirmed in
microcosm experiments (44).
Reseachers in Germany began a series of experiments in 1993 to monitor
field releases of transgenic rizomania-resistant sugar beet (Beta
vulgaris), containing the marker gene for kanamycin resistance, for
persistence of transgenic DNA and of horizontal gene transfer of
transgenic DNA into soil bacteria (45). It is the first such experiment to
be carried out; after tens of thousands of field releases and tens of
millions of hectares have been planted with transgenic crops. It will be
useful to review their findings in detail.
Transgenic DNA was found to persist in the soil for up to two years
after the transgenic crop was planted. Though they did not comment on it,
the data showed that the proportion of kanamycin resistant bacteria in the
soil increased significantly between 1.5 and 2 years. Could it be due to
horizontal transfer of antibiotic resistance marker gene in the transgenic
DNA? Although none of 4000 colonies of soil bacteria isolated a
rather small number - was found to have taken up transgenic DNA by the
probes available, two out of seven samples of total bacterial DNA
yielded positive results after 18 months. This suggests that horizontal
gene transfer may have taken place, but the specific bacteria which have
taken up the transgenic DNA cannot be isolated as colonies. That is not
surprising as less than 1% of all the bacteria in the soil are culturable.
The authors were careful not to rule out transgenic DNA being adsorbed to
the surface of bacteria rather than being tranferred into the bacteria.
The researchers also carried out microcosm experiments to which total
transgenic sugar-beet DNA was added to non-sterile soil with its natural
complement of microorganisms. The intensity of the signal for transgenic
DNA decreased during the first days and subsequently increased. This may
be interpreted as a sign that the transgenic DNA has been taken up by
bacteria and become amplified as a result.
In parallel, soil samples were plated and the total bacterial lawn
allowed to grow for 4 days, after which DNA was extracted. Several
positive signals were found, "which might indicate uptake of
transgenic DNA by competent bacteria."
The authors were cautious not to claim conclusive results simply because
the specific bacteria carrying the transgenic DNA sequences were not
isolated. The results do show, however, that horizontal gene transfer may
have taken place both in the field and in the soil microcosm.
DNA is not broken down sufficiently rapidly in the gut either, which is
why transfer of transgenic DNA to microorganisms in the gut of bee larvae
would not be surprising. A genetically engineered plasmid was found to
have a 6 to 25% survival after 60 min. of exposure to human saliva. The
partially degraded plasmid DNA was capable of transforming Streptococcus
gordonii, one of the bacteria that normally live in the human mouth
and pharynx. The frequency of transformation dropped exponentially with
time of exposure to saliva, but it was still detectable after 10 minutes.
Human saliva actually contains factors that promote competence of resident
bacteria to become transformed by DNA (46).
Viral DNA fed to mice is found to reach white blood cells, spleen and
liver cells via the intestinal wall, to become incorporated into the mouse
cell genome (47). When fed to pregnant mice, the viral DNA ends up in
cells of the fetuses and the new born animals, suggesting that it has gone
through the placenta as well (48). The authors remark that "The
consequences of foreign DNA uptake for mutagenesis and oncogenesis have
not yet been investigated (49)." As already mentioned, recent
experiments in gene therapy leave little doubt that naked nucleic acid
constructs can readily enter mammalian cells and in many cases become
incorporated into the cells genome.
Horizontal gene transfer is an established phenomenon. It has taken
place in our evolutionary past and is continuing today. All the signs are
that natural horizontal gene transfer is a regulated process, limited by
species barriers and by mechanisms that break down and inactivate foreign
genetic material. Unfortunately, genetic engineering has created a huge
variety of artificial constructs designed to cross all species barriers
and to invade essentially all genomes. Although the basic constructs are
the same for all applications, some of the most dangerous may be coming
from the waste disposal of contained users of transgenic organisms(50).
These will include constructs containing cancer genes from viruses and
cells from laboratories researching and developing cancer and cancer
drugs, virulence genes from bacteria and viruses in pathology labs. In
short, the biosphere is being exposed to all kinds of novel constructs
and gene combinations that did not previously exist in nature, and may
never have come into being but for genetic engineering.
There is an urgent need to establish effective regulatory oversight, in
the first instance, to prevent the escape and release of these dangerous
constructs into the environment, and then to consider whether some of the
most dangerous experiments should be allowed to continue at all.
- Thanks to Dr. Beatrix Tappeser, Institute for Applied Ecology,
Postfach 6226, D-79038, Freiburg, for this information. See also
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- See Ho, et al, 1998 (note 4); Ho et al, 2000 (note