SLIPPING THROUGH THE REGULATORY NET: Naked' and 'free' nucleic acids

Item

Title
SLIPPING THROUGH THE
REGULATORY NET:
Naked' and 'free' nucleic acids
extracted text
TWN Biotechnology & Biosafety Series
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SLIPPING
THROUGH THE
REGULATORY NET:
'Naked' and
'free' nucleic acids

by Mae-Wan Ho, Angela Ryan,
Joe Cummins & Terje Traavik

TWN

Third World Network

5

SLIPPING THROUGH THE
REGULATORY NET:
Naked' and 'free' nucleic acids

Mae-Wan Ho, Angela Ryan,
Joe Cummins & Terje Traavik

TWN
Third World Network
Penang, Malaysia

SLIPPING THROUGH THE REGULATORY NET:
'Naked' and 'free' nucleic acids
is published by
Third World Network
228 Macalister Rond
10400 Penang, Malaysia.

copyright © Third World Network 2001

Printed by Jutaprint
2 Solok Sungei Pinang 3, Sg. Pinang
11600 Penang, Malaysia.

ISBN: 983-9747-74-6

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Contents
Abstract

Chapter 1.

'Naked' and 'free' nucleic acids

1

Naked nucleic acids in genetic engineering
biotechnology
Free nucleic acids resulting from genetic
engineering biotechnology

4

Chapter 2.

DNA persists in all environments

6

Chapter 3.

The potential hazards of naked nucleic
acids

8

Hazards of naked nucleic acids

8

Chapter 4.

Chapter 5.

Chapter 6.

2

The horizontal transfer of transgenic DNA 11
Reasons to suspect that transgenic DNA
may be more likely to spread horizontally than
non-transgenic DNA

13

The hazards of horizontal gene transfer

14

Potential hazards from horizontal gene transfer of
naked/free nucleic acids

15

Conclusion

17

Endnotes

18

Appendix

24

Abstract
Biotechnological processes are creating an increasing variety of
naked/free nucleic acids that are released unregulated into the
environment. They range from oligonucleotides to artificial con­
structs thousands and millions of base-pairs in length, often con­
taining heterogeneous combinations of genes from pathogenic bac­
teria, viruses and other genetic parasites belonging to every king­
dom of living organisms. Most have never existed, or if they have,
not in such large amounts. They are, by definition, xenobiotics substances foreign to nature — with the potential to cause harm.
Nucleic acids are now known to persist in all environments, in­
cluding the digestive tract. Transformation by the uptake of DNA
is a significant route of horizontal gene transfer, and there is over­
whelming evidence that horizontal gene transfer and recombina­
tion have been responsible for the recent resurgence of drug and
antibiotic resistant infectious diseases.

Research findings in gene therapy and vaccine development show
that naked/free nucleic acids constructs are readily taken up by
the cells of all species including human beings. These nucleic acid
constructs can become integrated into the cell's genome. Integra­
tion may result in harmful biological effects, including cancers.
Unfortunately, regulation has lagged far behind scientific evidence.
The most serious is with regard to 'contained use'. Industrial con­
tained users release large volumes of untreated transgenic micro­
organisms, which they judge to be 'safe', directly into the environ­
ment. Moreover, transgenic wastes consisting of killed microorgan­
isms but containing large amounts of transgenic nucleic acids are being
recycled as food, feed and fertilizer, or disposed of in landfills. These prac­
tices are precisely those that can enhance horizontal transfer and
recombination of transgenic nucleic acids, and should no longer be
permitted.

Chapter 1

"Naked7 and 'free' nucleic acids

'Naked' nucleic acids are DNA/RNA produced in the laboratory
for use in, or as the result of, genetic engineering.

'Free' nucleic acids refer to these laboratory-produced nucleic ac­
ids transfected into cells or organisms, whether incorporated as
transgenic DNA or not, and subsequently released into the envi­
ronment by secretion, excretion, waste disposal, death, industrial
processing, or carried by liquid streams, or in airborne aerosol, dust
and pollen.
Genetic engineering is a valuable research tool, which may have
beneficial applications. But the continued research and develop­
ment of the technology must go hand in hand with appropriate
regulation in order that the benefits should not be outweighed by
the harm it may cause.
The naked nucleic acids produced in the laboratory (see Box 1)
range from oligonucleotides of less than 20 nucleotides to artificial
chromosomes millions of base-pairs in length. The constructs typi­
cally contain antibiotic resistance marker genes plus a heterogene­
ous combination of genes from pathogenic bacteria, viruses and
other genetic parasites belonging to every kingdom of living or­
ganisms on earth (1).

Most have never existed in nature, or if they have, not in such large
quantities. They are, by definition, xenobiotics - substances for-

1

RNA-based
Antisense RNA used in gene therapy
Ribozymes (RNA enzymes that cut RNA) used in gene therapy (4)
Self-replicating RNA (linked to RNA-dependent RNA polymerase)
used in gene therapy (5)
RNA vaccines
RNA viral vectors eg, alphaviruses, Venezuelan equine encephalitis vi­
rus

RNA-DNA hybrid
RNA-DNA hybrid sequence of 25 nucleotides used to introduce pre­
cise base-sequence changes in a target gene (6)

These constructs are therefore greatly amplified, and at the same
time, introduced into foreign genomes where recombination with
host genes and the genomes of the host's viral and bacterial patho­
gens may readily occur.

Transgenic wastes containing large amounts of free or potentially
free transgenic DNA are being released into the environment, in­
cluding those from microorganisms and cell cultures in 'contained
use' (see Box 2).
With the possible exception of viral genomes, viral vectors and
viroids (infectious naked RNA agents), neither naked nor free nu­
cleic acids fall within the scope of the current EC Directive (7) on
contained use and deliberate release.
They are also being excluded from the Cartegena Biosafety Proto­
col, an international agreement to regulate the international move­
ment, handling and use of GMOs, negotiated in Montreal in Janu­
ary 2000, and signed by more than 60 nations to date.

Worse still, current European regulation allows users to release
directly into the environment certain live transgenic microorgan-

3

eign to nature — with the potential to cause harm.

Many novel constructs are incorporated into transgenic microor­
ganisms, plant and animal cell cultures for commercial produc­
tions, and into crops, livestock, fish and other aquatic organisms
for food, animal feed, and other purposes.

Box 1: Naked nucleic acids in genetic engineering
biotechnology
DNA-bascd
Viral genomes, e.g., cauliflower mosaic virus, cytomegalovirus, vaccinia,
baculovirus, adenovirus, SV40, many bacteriophages
cDNA of RNA viral genomes, e.g., retroviruses, SIV, HIV, Rous Sar­
coma virus, mouse Moloney virus, Ebola virus
Plasmids, e.g., Ti of Agrobacterium, many plasmids from E. coli and
yeast, often carrying antibiotic resistance genes
Transposons, e.g., many broad host-range transposons from E. coli with
antibiotic resistance genes, some from Drosophila, such as mariner arc
found in all eukaryotic kingdoms
Artificial vectors made by recombining viral genomes, plasmids and
transposons, carrying antibiotic resistance genes, and used for gene am­
plification, DNA sequencing, transfection, gene therapy, etc., many are
shuttle-vectors designed for replication in more than one species, 'pan­
tropic' vectors cross many species barriers
Naked DNA vaccines, both plasmid-based and viral vector-based
Artificial chromosomes, yeast (YAC), plasmid (PAC) and mammalian
(MAC) (2)
Artificial constructs, transgene cassettes, often including antibiotic re­
sistance gene cassettes, with promoters from viruses, or synthetic su­
per-promoters and terminators
PCR amplified sequences
Oligodeoxynucleotides (antisense), hairpin-forming oligonucleotides
used in gene therapy (3)

2

RNA-bascd
Antisense RNA used in gene therapy
Ribozymes (RNA enzymes that cut RNA) used in gene therapy (4)
Self-replicating RNA (linked to RNA-dependent RNA polymerase)
used in gene therapy (5)
RNA vaccines
RNA viral vectors eg, alphaviruses, Venezuelan equine encephalitis vi­
rus
RNA-DNA hybrid
RNA-DNA hybrid sequence of 25 nucleotides used to introduce pre­
cise base-sequence changes in a target gene (6)

These constructs are therefore greatly amplified, and at the same
time, introduced into foreign genomes where recombination with
host genes and the genomes of the host's viral and bacterial patho­
gens may readily occur.

Transgenic wastes containing large amounts of free or potentially
free transgenic DNA are being released into the environment, in­
cluding those from microorganisms and cell cultures in 'contained
use' (see Box 2).
With the possible exception of viral genomes, viral vectors and
viroids (infectious naked RNA agents), neither naked nor free nu­
cleic acids fall within the scope of the current EC Directive (7) on
contained use and deliberate release.

They are also being excluded from the Cartegena Biosafety Proto­
col, an international agreement to regulate the international move­
ment, handling and use of GMOs, negotiated in Montreal in Janu­
ary 2000, and signed by more than 60 nations to date.

Worse still, current European regulation allows users to release
directly into the environment certain live transgenic microorgan-

3

Box 2: Free nucleic acids resulting from genetic
engineering biotechnology
Transfected, unincorporated nucleic acids/constructs due to gene
therapy, vaccination, and transgenesis, released into the environment
by secretion, excretion, waste carcass disposal, cell death, etc.
Transgenic DNA released from live or dead cells in the following:
• Transgenic wastes from genetically engineered microorganisms in
contained use
• Transgenic wastes from cell cultures in contained use
• Transgenic wastes from genetically engineered crops
• Transgenic wastes from genetically engineered fish and other aquatic
organisms
• Transgenic wastes from genetically engineered farm animals
• Unprocessed transgenic food and animal feed
• Processed transgenic food for human use and animal feed
• Processed transgenic textiles such as cotton
• Transgenic dust from food processing
• Transgenic pollen

isms considered nonpathogenic or otherwise safe in liquid waste,
although there is no agreement across European countries as to
which bacteria are pathogens (8) (see Appendix).
Meanwhile, all killed microorganisms and cells containing transgenic
DNA are disposed of as solid waste, and are either recycled as food, feed
and fertilizer, or disposed of in landfill and incineration, according to a
recent paper in the 'safe biotechnology' series produced by the
European Federation of Biotechnology (9).

The paper from industry considered the DNA content of biotech­
nological waste, and reaffirmed the adequacy of existing practice,
except "in cases where recombinant DNA is specifically constructed

4

to transform higher cells, such as gene vaccines or genetic-pill ap­
plications", where "it will be necessary to inactivate waste by vali­
dated procedures rendering DNA nonfunctional by either reduc­
ing DNA fragment size below functional entities or altering the
chemical composition and structure of the DNA."
At present, the 'validated procedure' for treating viral genomes
and viral vectors appears to involve no more than autoclaving, but
whether this sufficiently degrades nucleic acids is not known.

Historically, the lack of regulation of naked/free nucleic acids is
due largely to the assumption, now proven to be erroneous, that they
would be rapidly broken down in the environment and in the di­
gestive tract of animals (see the next chapter). Another assumption
is that as DNA is present in all organisms, it is not a hazardous
chemical, and hence there is no need to regulate it as such (10).

5

Chapter 2

DNA persists in all environments

Naked/free DNA is now known to persist in all natural environ­
ments. High concentrations of DNA are found in the soil, in ma­
rine and freshwater sediments and in the air-water interface, where
it retains the ability to transform microorganisms (11,12).

DNA also persists in the digestive tract (13), where sizable frag­
ments may be taken up and incorporated by resident microbes and
cells of the mammalian host.
A genetically engineered plasmid was found to have a 6% to 25%
survival after 60 minutes of exposure to human saliva (14). The
partially degraded DNA was capable of transforming Streptococ­
cus gordonii, one of the bacteria that normally live in the human
mouth and pharynx.
It has long been assumed that DNA cannot be taken up through
intact skin, surface wounds, or the intestinal tract, or that it would
be rapidly destroyed if taken up. Those assumptions have been
overtaken by empirical findings. The ability of naked DNA to pen­
etrate intact skin has been known at least since 1990.

Cancer researchers found that within weeks of applying the cloned
DNA of a human oncogene to the skin on the back of mice, tu­
mours developed in endothelial cells lining the blood vessels and
lymph nodes (15).

6

Viral DNA fed to mice reached white blood cells, spleen and liver
cells via the intestinal wall, to become incorporated into the mouse
cell genome (16). When fed to pregnant mice, the viral DNA ended
up in cells of the fetuses and the newborn, indicating that it had
gone through the placenta (17).

Recent research in gene therapy shows how readily naked nucleic
acids can enter practically every type of human cells and cells of
model mammals.
Naked nucleic acids can be successfully delivered, either alone or
in complex with liposomes and other carriers, in aerosols via the
respiratory tract (18), by topical application to the eye (19), to the
inner ear (20), to hair follicles by rubbing on the skin (21), by direct
injection into muscle (22), through the skin (23), and by mouth,
where the nucleic acid is taken up by cells lining the gut (24).

Naked DNA can even be taken up by sperms of marine organisms
and mammals, and transgenic animals created (25). Researchers
have also found unintended integration as, for example, of a plas­
mid-based naked DNA malarial vaccine injected into mouse mus­
cle (26).

7

Chapter 3

The potential hazards of naked nucleic
acids

The potential hazards of naked nucleic acids are summarised in
Box 3.

Box 3: Hazards of naked nucleic acids
• Acute toxic shock from viral vectors
• Immunological reaction from viral vectors
• Autoimmune reactions from double-stranded DNA and RNA
• Non-target interference with gene function from antisense DNA,
RNA and ribozymes
• Generation of virulent recombinant viruses
• Insertion mutagenesis
• Insertion oncogenesis
• Genetic contamination of germ cells

Naked viral genomes often have a wider host range than the intact
virus. Human T-cell leukaemia viral genomes formed complete
viruses (27), and naked genomes from the human polyomavirus
BK (BKV) gave a full-blown infection when injected into rabbits,
despite the fact that neither intact virus is infectious for rabbits
(28). This is particularly relevant to the virus-based gene therapy
vectors and naked DNA vaccines under development (29).

It is already known that even small modifications to viral genomes,
such as insertions, deletions and single nucleotide substitutions,
can have unexpected effects on virulence and host range (30). Re­

8

searchers In Canberra, Australia, accidentally created a deadly
transgenic virus that killed everyone of its victims simply by in­
serting the gene for interleukin-4 into the relatively harmless mouse­
pox virus (31).
Gene-therapy vectors and naked DNA vaccines have caused acute
toxic shock reactions (32) and severe immune reactions (33). Be­
tween 1998 and 1999, scientists in US drug companies failed to
notify the regulatory authorities of six deaths and more than 650
adverse events resulting from clinical trials of gene therapy, the
precise causes of which are yet to be determined (34).

Naked DNA can also trigger autoimmune reactions. Any fragment
of double-stranded DNA or RNA (down to 25 base-pairs) intro­
duced into cells can induce those reactions, which are linked to
rheumatoid arthritis, insuiin-dependent diabetes and Graves dis­
ease of the thyroid (35). Double-stranded RNA mainly appears
during viral infection, and is recognized as a trigger for activating
genes that produce interferons (36).
Many 'spontaneous' mutations result from insertions of
transposons and other invasive elements. Insertion mutagenesis is
associated with a range of cancers of the lung (37), breast (38), co­
lon (39) and liver (40). Finally, unintended modification of germ­
cells can result from gene therapy and vaccinations (25).
Naked RNA genomes are also infectious. Viroids (41) and viroidlike satellite RNAs (42) from plants and the hepatitis 8 virus (43) in
humans are the smallest known agents of infectious disease. They
are single-stranded RNA molecules that lack both protein coat and
detectable mRNA activity.

Antisense RNA, like antisense DNA, will be expected to have bio­
logical effects either in blocking the function of homologous genes
or genes with homologous domains.

9

RNA may also be reverse-transcribed into complementary DNA
(cDNA) by reverse transcriptase, which is present in all higher or­
ganisms (44) and in some bacteria (45); the cDNA may then be­
come incorporated into the cell's genome.

The direct uptake and incorporation of genetic material from unre­
lated species is referred to as horizontal gene transfer, as distinct from
the usual vertical gene transfer from parent to offspring in repro­
duction.

10

Chapter 4

The horizontal transfer of transgenic DNA

Many geneticists accept that naked nucleic acids are transferred
horizontally, especially to microorganisms, but dispute the trans­
fer of transgenic DNA, which they regard to be no different from
host-cell DNA. But there is already evidence of horizontal transfer
of plant transgenic DNA; and there is no reason to believe that the
same may not apply to transgenic DNA in animals - fish, labora­
tory mice and rats, livestock and insects - as all transgenic con­
structs are similar.

Secondary horizontal transfer of transgenic DNA to soil bacteria
and fungi has been demonstrated in the laboratory. In the case of
fungi, the transfer was obtained simply by co-cultivation (46). Suc­
cessful transfers of a kanamycin resistance marker gene to the soil
bacterium Acinetobacter were obtained using DNA extracted from
homogenized plant leaf from a wide range of transgenic plants:
potato, tobacco, sugar beet, oil-seed rape and tomato (47). About
2,500 copies of the kanamycin resistance genes (from the same
number of plant cells) are sufficient to successfully transform one
bacterium, despite the six million-fold excess of plant DNA present.
There are claims that horizontal gene transfer has close to zero prob­
ability of occurring in nature. But this is based more on assump­
tions than experimental investigations under realistic conditions.
Gebbard and Smalla (48) have found horizontal transfer of kan­
amycin resistance genes from transgenic DNA to Acinetobacter, and

11

positive results were obtained using just 100j.il of plant-leaf homoge­
nate.

Horizontal transfer of transgenic DNA has actually been found in
the field. Transgenic DNA was detected in the soil at least two years
after the transgenic sugarbeet crop was harvested. Specific PCR
probes show that different parts of the transgenic DNA may have
been taken up by soil bacteria, although the bacterial strains them­
selves could not be isolated (49); which is not surprising as less
than 1% of soil bacteria can be isolated.
There are also reasons to suspect that transgenic DNA may be more
likely to transfer horizontally than the organism's own genes (see
Box 4). Natural genetic material, say, in non-GM food, is generally
broken down to provide energy and building-blocks for growth
and repair. And in the rare event that the foreign genetic material
gets into a cell's genome, other mechanisms can still put the for­
eign genes out of action or eliminate it. These are all part of the
biological barrier that keeps species distinct, so gene exchange
across species is held in check.

Almost by definition, genetic engineering involves designing arti­
ficial constructs to invade genomes and to overcome natural spe­
cies barriers. Because of their highly mixed origins, transgenic con­
structs tend to be unstable as well as invasive, and may therefore
be more likely to spread by horizontal gene transfer.

12

Box 4: Reasons to suspect that transgenic DNA
may be more likely to spread horizontally than
non-transgenic DNA
1. Artificial constructs and vectors are designed to be invasive to for­
eign genomes and overcome species barriers.
2. Artificial gene constructs tend to be structurally unstable (50, 51),
and hence more prone to recombine and transfer horizontally.
3. The mechanisms enabling foreign genes to insert into the genome
also enable them to jump out again, to reinsert at another site, or to
another genome.
4. The integration sites of most commonly used artificial vectors for
transferring genes are 'recombination hotspots', and so have an increased
propensity to transfer horizontally.
5. Viral promoters, such as that from the cauliflower mosaic virus,
widely used to make transgenes over-express, contain recombination
hotspots (52), and will therefore further enhance horizontal gene trans­
fer.
6. The metabolic stress on the host organism due to the continuous
over-expression of transgenes may also contribute to the instability of
the insert (53).
7. The foreign gene constructs, and the vectors into which they are
spliced, are typically mosaics of DNA sequences from numerous spe­
cies 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.

13

Chapter 5

The hazards of horizontal gene transfer

Among the scientific advice given by the UK Ministry of Agricul­
ture, Fisheries and Food (MAFF) to the US Food and Drug Admin­
istration (FDA) (54) at the end of 1998 are the following warnings:
1. Transgenic DNA can spread to farm workers and food proces­
sors via dust and pollen.
2. Antibiotic resistance marker genes may spread to bacteria in
the mouth, as the mouth contains bacteria that readily take up and
incorporate foreign DNA. Similar transformable bacteria are present
in the respiratory tracts.
3. Antibiotic resistance marker genes may spread to bacteria in
the environment, which then serves as a reservoir for antibiotic
resistance genes.
4. DNA is not readily degraded during food processing nor in the
silage (55), hence transgenic DNA in animal feed can spread to
bacteria in animals.
5. Foreign DNA can be delivered into mammalian cells by bacte­
ria that can enter the cells (56).
6. The ampicillin resistance gene in the transgenic maize under­
going 'farm-scale' field-trials in the UK and elsewhere is very mu­
table, and may compromise treatment for meningitis and other bac­
terial infections, should the gene be transferred horizontally to the
bacteria.

The potential hazards of horizontal gene transfer are unlike those
of any other process (see Box 5).

14

Box 5: Potential hazards from horizontal gene
transfer of naked/free nucleic acids
• Generation of new 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 most, if not all, cells
and genomes, which may cause diseases
• Multiplication of ecological impacts due to all the above

There is already overwhelming evidence that horizontal gene trans­
fer has been responsible for creating new viral and bacterial patho­
gens and for spreading drug and antibiotic resistance among the
pathogens (1).
Thus, it can be expected that a technology that enhances horizon­
tal gene transfer, both by design and otherwise, will also contrib­
ute to creating new pathogens and to spreading drug and antibi­
otic resistance. We have already referred to the evidence for inser­
tion mutagenesis earlier. The potential for reactivating dormant
viruses cannot be dismissed.
We have drawn attention recently to the cauliflower mosaic virus
(CaMV) 35 S promoter (57-60) widely used to make transgenes over­
express constitutively, and is in practically all transgenic crops al­
ready released commercially or undergoing field trials.
This promoter not only has a recombination hotspot (see Chapter
4), but is completely promiscuous in that it functions efficiently in
all plants, green algae, yeast and E. coli, as well as animal and hu­
man cells. It has a modular structure, with parts common to, and
15

interchangeable with promoters of other plant and animal viruses.
Dormant and relict viral sequences have been discovered in the
human and other animal genomes at least 20 years ago (61). Proviral sequences and related retrotransposons have also been discov­
ered recently in plant genomes (62).
Proviral sequences in animal genomes can be reactivated, most
strikingly, in the phenomenon of xenotropism in which they be­
come infectious for cells for other species (63), constituting a major
safety issue for xenotransplantation. Plant proviral sequences can
also be activated, in some cases simply during plant tissue culture
(64).

Recombination between promoters is not a theoretical possibility
any more than the reactivation of dormant viruses. Synthetic su­
per-promoters for gene therapy have been created in the labora­
tory by random recombination of modules isolated from natural
promoters (65).
In gene therapy, a major safety concern is, indeed, the generation
of 'replication competent viruses' (RCV) due to recombination of
viral vectors with proviral and other sequences in the genomes of
cell-lines used to package the viral vectors (66).

16

Chapter 6

Conclusion

The naked/free nucleic acids created by genetic engineering bio­
technology are potentially the most hazardous xenobiotics to pol­
lute our environment.

Unlike chemical pollutants that dilute out and degrade over time,
nucleic acids can be taken up by all cells to multiply, mutate and
recombine indefinitely.
There is an urgent need to take appropriate measures to prevent
the release of any of these naked/free nucleic acids into the envi­
ronment.

In particular, the practices of recycling solid transgenic wastes as
food, feed, fertilizer and landfills by the biotech industry should
no longer be permitted until and unless they are demonstrated to
be safe beyond reasonable doubt as the result of dedicated empiri­
cal investigations.

17

Endnotes
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3. Helin V, Gottikh M, Mishal Z, Subra F, Malvy C and Lavignon M.
Cell cycle-dependent distribution and specific inhibitory effect of
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macology 1999: 58: 95-107.
4. Hammann C and Tabler M. Generation and application of asymmet­
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7. Council Directive 90/219/EEC on contained use of genetically modi­
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05.12.1998) and Council Directive on the deliberate release into the
environment of genetically modified organisms 90/220/EEC, last
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Executive when asked whether there was any recommended treat­
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1.

18

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19

25.

Spadafora C. Sperm cells and foreign DNA: a controversial relation.
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Appendix
How the EC Directive on Contained Use Allows Dangerous GM Wastes
to be Recycled as Food Feed and Fertilizer
The safety of GM crops and foods has been grabbing headlines over the
past two years. However, a potentially much more serious source of haz­
ard remains unregulated. We first pointed to fundamental flaws in the
regulation on contained use in a comprehensive review published in a
scientific journal in 1998 [1]. This paper was submitted to the World Health
Organization, European Commission, the Biosafety Conferences at the
UN, as well as to the UK Health and Safety Executive, with additional
comments from Mae-Wan Ho and others.

More recently, we raised the matter again in an update calling attention
to the increasing variety and volume of 'naked' and 'free' nucleic acids
produced in the laboratory and biotech factories under contained use,
that are in fact not contained at all, but discharged in one form or another
into the environment, as sanctioned by the current EC Directive on Con­
tained Use (Council Directive 90/219/EEC), last amended in 1998. Our
paper was circulated at the Montreal meeting on Biosafety in January.
But there has been no real change since to the Directive on Contained
Use. This Directive is fundamentally inadequate for the following rea­
sons:
•

The scope covers only genetically modified microorganisms;
transgenic animals, fish and plants are not included. It also excludes
nearly all classes of naked or free nucleic acids, except for viroids
(infectious naked RNAs that cause diseases in both plants and ani­
mals).

•

Notification only and not explicit approval is needed for use of Group
1 GM microorganisms, GMMs, considered nonpathogenic or other­
wise safe; however, there is no agreement among EU nations on which
microorganisms are pathogens or not; and it is effectively left up to
industry to decide.

24

For Group 1 GMMs, only 'principles of good microbiological prac­
tice' applies, that is, there is no containment.
•

'Tolerated release' of Group 1 GMMs are allowed to take place, with­
out treatment, directly into the environment.

•

No treatment of GM DNA or RNA is required to break them down
fully before release.

•

There is no requirement to monitor for escape of GMMs or GM con­
structs, horizontal gene transfer, or impacts on health and biodiversity.

We have presented evidence alerting to the dangers of horizontal gene
transfer, among which are the creation of new viral and bacterial patho­
gens and the spread of antibiotic and drug resistance among the patho­
gens.
Despite our efforts, successive versions of the Directive have been relaxed
and shaped by the European Federation of Biotechnology. This industrydominated group have produced a series of 'safe biotechnology' papers,
one of which [9] specifically addresses DNA content of biotechnological
wastes.

The paper admits that DNA persists in soil and aqueous environments,
that it is transferred to bacteria and cells of animals, and that it may be­
come integrated into their genomes.
But they defend current practice by claiming

1)

horizontal transfer of GM DNA occurs, if at all, at very low frequen­
cies, especially in nature,

2)

the persistence of foreign DNA depends on selective pressure, espe­
cially in the case of antibiotic resistance marker genes, and

3)

DNA taken up is unlikely to be integrated into the cell's genome un­
less designed to do so.

25

The first claim is unwarranted. Evidence of horizontal gene transfer from
transgenic plants to soil bacteria has been obtained in the laboratory as
well as in the field, although the researchers themselves are downplaying
the findings, in violation of the precautionary principle [see ref. 67],

The second assumption has been shown to be false. There is now sub­
stantial evidence that antibiotic resistance can and does persist in the ab­
sence of the antibiotic [68], mainly because biological functions are, as a
rule, all tangled up with one another, and cannot be neatly separated and
selected one at a time.

The third point is false as well, for it has been demonstrated in gene'therapy' experiments (see main text) that naked DNA constructs, not in­
tended for integration, have nevertheless become integrated into the ge­
nome. Integration occurs not only in somatic cells, but also in germ-cells.
The most dangerous aspect of current practice, defended by industry, is
that solid wastes, heat-treated, or autoclaved, containing large amounts
of intact or incompletely degraded GM constructs and transgenic DNA
are being recycled or disposed of as food, feed, fertilizer, land reclama­
tion and landfill.

Only in cases where GM constructs are specifically made to transform
higher organisms, such as gene vaccines and genetic-pill applications (for
gene therapy) is there a recognition that there may be a need to 'inacti­
vate waste by validated procedures rendering DNA nonfunctional by ei­
ther reducing DNA fragment size below functional entities or altering
the chemical composition and structure of the DNA.' However, no such
validated procedures exist.
Our regulatory authorities at all levels persist in ignoring scientific ad­
vice and scientific evidence. It is an example of the anti-precautionary
approach [69], They, together with the biotech industry, should be held
legally responsible for any harm resulting from the uncontrollable hori­
zontal transfer and recombination of GM material.

26

Biotechnological processes are creating an increasing variety of
naked/free nucleic acids that are released unregulated into (and which
persist in) the environment. They are by definition, xenobiotics substances foreign to nature. Research findings in gene therapy and
vaccine development show that naked/free nucleic acids constructs
are readily taken up by the cells of all species including human
beings. These nucleic acid constructs can become integrated into
the cell’s genome and such integration may result in harmful bio­
logical effects, including cancers.
Of current and serious concern is with regard to ‘contained use’.
Industrial contained users release large volumes of untreated
transgenic microorganisms, which they judge to be ‘safe’, directly
into the environment. Moreover, transgenic wastes consisting of
killed microorganisms but containing large amounts of transgenic
nucleic acids, are being recycled as food, feed and fertilizer, or dis­
posed of in landfills. These practices are precisely those that can
enhance horizontal transfer and recombination of transgenic nu­
cleic acids, and should no longer be permitted.
Dr Mae-Wan Ho is Senior Research Fellow of the Open University, UK, Fellow
of the US National Genetics Foundation, and co-founder and director of the
institute of Science in Society (ISIS).
Angela Ryan read Molecular Biology at Kings College, London, was research

Fellow in molecular biology at the Open University for ISIS and is undertaking
a Masters in Science, Culture and the Environment at the University of London.
Dr Joe Cummins is Professor Emeritus of Genetics at the University of West­
ern Ontario, Canada. He has a PhD degree in Cellular Biology and has taught
genetics at several universities in the USA.
Dr Terje Traavik is Professor and Head of the Department of Virology, at the

Institute of Medical Biology, University of Tromso, Norway and is co-founder
and scientific director of GENOK-Norwegian Institute of Gene Ecology.
BIOTECHNOLOGY & BIOSAFETY SERIES
is a series of papers published by the Third World Network. It is
aimed at deepening public understanding of ecological and safety
aspects of new biotechnologies, especially genetic engineering.
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