"Genetically Modified Plants and the 35S Promoter: Assessing the
Risks and Enhancing the Debate"
R. Hull, S.N. Covey and P. Dale
John Innes Centre
Norwich Research Park
Colney, Norwich NR4 7UH, UK
Microb. Ecol. Health Dis.
The 35S promoter, derived from the common plant virus, cauliflower mosaic
virus (CaMV), is a component of transgenic constructs in more than 80% of
genetically modified (GM) plants. Alarming reports have suggested that the
35S promoter might cause accidental activation of plant genes or endogenous
viruses, promote horizontal gene transfer, or might even recombine with
mammalian viruses such as HIV, with unexpected consequences. In this
article, we discuss the properties of CaMV and the 35S promoter and the
potential risks associated with the use of the promoter in GM plants,
concluding that any risks are no greater than those encountered in
conventional plant breeding.
In a recent article, Ho et al. (1999) suggested that the widespread use of
the 35S promoter of cauliflower mosaic virus (CaMV) in transgenic plants is
"a recipe for disaster". Ho et al. (1999) base their arguments on three
considerations, a) that the CaMV 35S promoter has a hotspot for
recombination (Kohli et al., 1999); b) that the 35S promoter has several
domains with different tissue specificities and c) that the 35S promoter is
very efficient and can function in a wide range of organisms, not only
plants but also bacteria and animals. From this they deduce that the 35S
promoter could recombine to activate dormant viruses, create new viruses and
"cause cancer by the overexpression of normal genes". As scientists who
have worked on CaMV for up to 25 years and have contributed much to the
understanding of its molecular biology we wish to put these scenarios in the
The first plant promoter
Over 15 years ago, CaMV was one of several plant genetic systems being
studied for its potential use in plant transformation (Hull, 1983; 1984;
1985). As part of these studies, much basic research went on into
understanding the genetic organisation of CaMV and the means by which its
genes are expressed and regulated. The CaMV genome was the first
significant piece of plant DNA to be completely sequenced (Frank et al.,
1980) and the two CaMV promoters, the 35S and 19S promoters, were the first
plant promoters identified (Covey et al., 1981; Hull and Covey, 1983c; Odell
et al., 1995). Because of the latter discovery, and the finding that the 35S
promoter was active in directing heterologous expression of plant genes in a
variety of plants, its use in the development of GM plants for research and
agronomic applications became widespread.
CaMV can infect a wide range of crucifers (see Schoelz and Bourque, 1999)
and is commonly found in cabbages, cauliflowers, oilseed rape, mustard and
other brassicas in temperate countries (Tomlinson, 1987). A survey of a
local market, as part of a risk assessment exercise for the Ministry of
Agriculture, Fisheries and Food (the UK regulatory authority for biosafety
of genetic manipulation of plant pests) in the late 1980s, showed that about
10% of the cauliflowers and cabbages were infected with CaMV. The virus is
transmitted in nature by aphids (see Schoelz and Bourque, 1999) and it might
be expected that organically grown crucifers, on which the aphids have not
been controlled by insecticides, would have higher rates of infection.
Infection early in the plant growth might affect the quality, especially of
cauliflowers, but later infections show leaf symptoms but little other overt
effects. The virus infects most cells of the plant and produces about 105
particles per cell. Each particle contains one molecule of the viral
genome, an 8 kbp circular double-stranded DNA with one copy each of the two
The replication cycle of the virus has two phases (see Hohn, 1999), the
first in the nucleus where the viral genome is uncoated, forms a
minichromosome and is transcribed to give two RNA species, the 35S RNA
(using the 35S promoter) and the 19S RNA (using another promoter). These
RNAs pass to the cytoplasm where the 35S RNA acts as a template for reverse
transcription as well as a template for translation of some gene products;
the 19S RNA is the template for the translation of just one gene product.
Various unencapsidated replication intermediates are found in infected cells
(Hull and Covey, 1983a) which are estimated to give a further 104 CaMV
molecules per cell. CaMV was the first plant virus shown to involve
reverse transcription in its replication (Hull and Covey, 1983b; Pfeiffer
and Hohn, 1983) but has now been joined by more than a thirty other plant
viruses which have the same replication mechanism.
Reverse transcribing elements
There is a wide range of reverse transcribing elements from animals, plants,
bacteria and fungi (Hull, 1999). These elements are grouped together on
common features of replication which use the enzyme reverse transcriptase.
It is considered likely that they had a common ancestor. There are two basic
types of reverse transcribing elements. Reverse transcribing viruses express
gene products which enable them to move between hosts. These viruses fall
into two basic groups, the retroviruses which encapsidate RNA and whose
replication involves integration of the viral sequence into the host genome,
and pararetroviruses which encapsidate DNA and whose normal replication
does not involve integration of the viral sequence. In contrast,
retroelements are reverse transcribing sequences integrated into the host
genome but which do not produce gene products necessary for easy horizontal
movement between hosts.
The retrovirus family comprises a large number of viruses which infect
vertebrates and cause a range of diseases including tumors, leukemia and
immunodeficiency. Retroviruses express a gene product, the integrase
protein, which facilitates the integration of the viral genome into the host
chromosomes where it can stay dormant for a considerable time. The virus
particles, which are horizontally transmitted, contain RNA transcribed from
this integrated genome. On entry into a new host this RNA is reverse
transcribed to give the DNA which is then integrated.
There are two groups of plant pararetroviruses, the caulimoviruses and the
badnaviruses (Hull, 1995; Lockhart et al., 1995) (the International
Committee on Taxonomy of Viruses has a more complex classification system
but for this article it is best to consider these viruses in these two
groups). The caulimovirus group includes viruses which infect groundnuts,
soybeans and cassava as well as brassicas. Crops infected by badnaviruses
include banana, cocoa, citrus, yams, pineapple and sugarcane. Although
there are differences in genome organisation between these two virus groups
their replication and the expression of their genes are very similar. The
DNA in the virus particles is transcribed in the nucleus (using the 35S
promoter) to give RNA which is replicated by reverse transcription to form
the DNA molecules which are encapsidated in the virus particles.
The one group of animal pararetroviruses, the hepadnaviruses, contains the
human-infecting hepatitis B virus and several viruses which infect ground
squirrels, woodchucks and ducks. These viruses have a very different genome
organisation to plant pararetroviruses, and although they replicate by
reverse transcription, there are major differences from retroviruses and
plant pararetroviruses in the details of the replication mechanism ( Mason
et al., 1987; compare Seeger 1999 for hepadnaviruses with Hohn, 1999 for
Retrotransposons are similar to animal retroviruses in that they are
integrated into the host chromosomes and their replication is by mechanisms
basically the same as those of retroviruses using a promoter analogous to
the 35S promoter. As noted above, they lack the gene products which enable
them to spread horizontally between hosts as do the retroviruses. However,
there is some recent evidence that they can move very infrequently between
hosts (Jordan et al., 1999) by an unknown mechanism. Plant genomes contain
large numbers of retrotransposons (Bennetzen and Kellog, 1997). For
instance, up to 50% of the maize genome is made up of retrotransposons
(SanMiguel et al., 1996) though many of these have mutated and are likely to
Relationships between reverse transcribing elements
As noted above, all these elements have a common mode of replication
involving reverse transcription of RNA to form DNA. However, functionally
reverse transcribing elements fall into two types, the retroviruses, plant
pararetroviruses and retrotransposons forming one type and the animal
pararetroviruses forming the other type. Within these types, each virus or
element is a highly coordinated structure and perturbation of the sequence
leads to loss of infectivity or functionality. This is exemplified by the
attempts to use cauliflower mosaic virus as a gene vector (see Hull, 1983).
In most cases, it is only when closely related sequences are added to or
exchanged within the genome that viability is retained.
There are many instances of plant and animal cells containing more than one
type of reverse transcribing virus or element. For instance, there are
several different human retroviruses and no instance of recombination has
been found between them. This indicates that there are many constraints on
Now to consider the specific points raised by Ho et al. (1999).
- The integrated 35S promoter can recombine with dormant viruses and also
create new viruses. We need to consider the situation firstly in plants and
then in animals which may eat those plants
- As noted above, there are more than 105 copies of the 35S promoter in
each cell of a plant naturally infected by CaMV, in contrast to the one to a
few copies of the 35S promoter in each cell of transformed plants. All
these other pararetroviruses have similar numbers of copies of their genomes
in their hosts but differ markedly in sequence from CaMV, even in the 35S
promoter region. Also noted above is that plants contain large numbers of
retrotransposons. In spite of these high numbers of both 35S promoter and
retrotransposons no cases of natural recombination leading to new viruses
have been found in spite of intensive research on these virus groups.
- There is uncertainty concerning the stage of transformation at which the
recombination described by Kohli et al. (1999) occurred. They did not
distinguish between recombination taking place during the process of
transformation and recombination taking place after the sequences had been
integrated. There is accumulating evidence of rearrangements of DNA during
transformation (e.g. De Groot et al., 1994; Register et al., 1994). In most
cases, these rearrangements result in the non-functioning of the transgene
and are selected out in the early stages of analysis of the properties of
the transformed lines. Furthermore, the construct used for transformation
by Kohli et al (1999) had three copies of the 35S promoter, one in inverse
orientation in relation to the other two. The presence of repeated
sequences in transformed integrants, and especially inverse repeats, also
tends to lead to gene silencing (see Kooter et al. 1999), a condition which
would be selected against in developing the transgenic line.
- Assuming that the integrated 35S promoter does have recombinational
properties, for it to effect the activation of a dormant virus or create a
new virus, the whole promoter would have to be either excised and reinserted
precisely at the new site or its 3' end linked precisely with another gene.
The first case means that there would have to be two recombination
"hotspots", that identified by Kohli et al. (1999) just downstream of the
TATA box and an upstream one which would enable the other promoter elements
to be included in the excised fragment. There is no evidence for such an
upstream "hotspot". Excision of the promoter would also be required for any
potential effects on animals.
It is important to note that genetic recombination is a normal feature of
conventional plant breeding and of all natural populations (Hayward et al.,
1993). Genetic variation is obtained by recombination following
hybridisation between genetically different plants which may be from
different species and genera. There are also irregular sources of
recombination by minor and major chromosomal rearrangements and from the
movement of transposable elements, which can move from one part of the
genome to another. Thus, recombination and hotspots for recombination are
not unique features of the CaMV 35S promoter
- The scenario suggested here by Ho et al. (1999) is that the 35S promoter
would recombine with hepadnaviruses such as human hepatitis B virus. This
is based on the suggestion of a "close relationship between CaMV and
hepadnaviruses". Hepadnaviruses have been classed as pararetroviruses as
they have a DNA genome and they do not involve integration in their
replication cycle. However, as noted above, their replication cycle differs
significantly from that of caulimoviruses. Furthermore, there is no
sequence similarity between the "hotspot" on the CaMV 35S promoter and
hepadnavirus sequences important in replication.
- Various problems would have to be overcome for the CaMV 35S promoter in
transgenic food plants to interact with human (or other animal) viruses or
any other sequences. Firstly, as noted above, the promoter would have to
excise in an appropriate manner. If the transgenic food was cooked the DNA
would be denatured and be very unlikely to renature in an operable form.
The next major problem is that the DNA containing the promoter would be
exposed to nucleases both from the plant cells when they were disrupted and
in the animals gut. Less than 5% administered DNA survives up to 7 hours in
an animal gut and that DNA is cleaved into very small pieces (Schubbert et
al., 1994). The DNA would then have to pass into the gut cells and
integrate precisely to activate the animal sequence. As noted above, the
consumption of CaMV-infected vegetables would result in the ingestion of
vastly more copies of the 35S promoter than the consumption of transgenic
plants containing the promoter. Brassicas are not the only crops which
contain pararetrovirus sequences. All banana varieties (and other Musa
varieties), the worlds fourth most important agricultural commodity, have
multiple copies of the sequences of banana streak badnavirus integrated into
their genomes ( Harper et al., 1999; Ndowora et al., 1999). In spite of
exposure of humans to these pararetroviruses there is no evidence of any ill
effects from them even in countries such as Uganda where bananas are the
staple diet and HIV is rife.
- That recombination of the 35S promoter would lead to overexpression of
Once again, one has to consider both plants and animals separately.
- The same arguments apply here as those under a) - c) above. However, if
we assume that the 35S promoter does recombine to enhance the expression of
a "normal" gene leading to the plant producing a harmful substance what are
the factors to be considered? Recombination is likely to be a very rare
event and to be in one or a few cells. For it to have any impact it would
have to be in germline cells so that it could be fully expressed in the
progeny. Also to have any impact it would have to be in an early generation
of bulking up of seed of that line. Recombination would result in the loss
of the transgenic character which would be recognised in these early
generations. By affecting a "normal" plant gene it would probably affect
the plant phenotype. Thus, the normal procedures used over many years for
the selection of new plant varieties from conventional breeding programmes
would identify this significant change.
- Plants contain many secondary metabolites which have evolved to provide
defense mechanisms against herbivores. The standard tests for carcinogens
using rodents indicate that a significant proportion of these secondary
metabolites are "carcinogens". For instance, out of 28 tested from coffee,
19 were carcinogenic in rodents and 35 out of 63 natural plant products were
carcinogens (Ames and Gold, 1997). Since these potentially carcinogenic
compounds have not been implicated in causing human cancers, it is
considered that humans are more adapted to them. Thus, the overexpression
of normal genes is very unlikely to cause cancers
- The same arguments as in d) and e) for the 35S promoter recombining with
an animal gene sequence also apply here. It must be remembered that animals
other than humans also eat brassicas and there are no reports of any
poisoning or disease which could be related to CaMV infection.
The paper by Ho et al. (1999) highlights some of the basic features of the
controversy over genetically modified crops. The transgenic situation has
to be compared with the natural situation not with a utopian one. It is
well known that viral sequences recombine naturally and that the vast
majority of these recombinants are unsuccessful. Very occasionally new
viruses arise, this being one of the major ways by which viruses evolve.
However, this recombination is between viruses which occur in plant cells at
very much higher concentrations than those of transgenic sequences, whether
they be promoters or the transgenes themselves. Furthermore, plants are
full of noxious substances which have evolved over many millenia to protect
the plant against herbivores and pathogens. Plant breeding and selection
has, and is, continuing to minimize(d) the effects that these substances
have on humans.
From the arguments above, there is no evidence that the CaMV 35S promoter
will increase the risk over those already existing from the breeding and
cultivation of conventional crops. . There is no evidence that the 35S
promoter, or other retroelement promoters, will have any direct effects, in
spite of being consumed in much larger quantities than would be from
transgenes in GM crops. Furthermore, there are compelling arguments to
support the view that there would be no more risks arising from potential
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