WESSEX
Campaign to ban genetically engineered food
WESSEX
Campaign to ban genetically engineered food
Risks Associated with the Use of the Cauliflower
Mosaic Virus Promoter in Transgenic Crops
( The address of this page is www.btinternet.com/~nlpwessex/Documents/camv.htm
)
The Cauliflower Mosaic Virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. It is potentially dangerous.
It is inserted into transgenic plants in a form which is different to its naturally ocurring state arising in its natural brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible.
This page provides more information on this specialist area of genetic engineering risk
The 35S CaMV plant virus promoter is active in human
enterocyte-like cells
European Food Research and Technology
Publisher: Springer-Verlag GmbH
ISSN: 1438-2377 (Paper) 1438-2385 (Online)
DOI: 10.1007/s00217-005-0154-3
Issue: Volume 222, Numbers 1-2
Date: January 2006
Pages: 185 - 193
"The 35S cauliflower mosaic virus (CaMV) promoter is
commonly used to drive transgene expression in the genetically engineered (GE) crop plants
that have been commercialized so far. Whether, and how far, the 35S promoter might be
active in mammalian cells has been scientifically unsettled and controversial...... We
constructed expression vectors with 35S promoter inserted in front of two reporter genes
encoding firefly luciferase and green fluorescent protein (GFP), respectively, and
performed transient transfection experiments in the human enterocyte-like cell line
Caco-2. It was demonstrated that the 35S CaMV promoter was able to drive the expression of
both reporter genes to significant levels...."
Click Here For More Information On The Above Paper
"At present, the success of transgene expression is variable, and many transformation experiments have to be carried out in order to isolate a small number of useful lines. Many factors can influence the behaviour of foreign DNA when it integrates into the plant genome. Such factors include the position of integration, possible rearrangements of the exogenous DNA by recombination, and the activation of endogenous plant defence systems that have evolved to suppress the activity of 'invading' DNA. (Fig.17)..."
John Innes Centre and Sainsbury Laboratory Annual Report 1998/99, p22-23
"Fig.17 Transgene rearrangements often occur at regions rich in DNA secondary structure, such as the CAMV 35S promoter, which can form the cruciform structure shown above. This allows recombination to occur, as shown by the green arrow."
John Innes Centre and Sainsbury Laboratory Annual Report 1998/99, p22 -23
Christou P, Kohli A, Stoger E, et al. 'Transgenic plants: a tool for fundamental genomics research'. John Innes Centre & Sainsbury Laboratory Annual Report 1999/2000, p.29.
Dr. Joseph Cummins, Professor Emeritus in genetics from the university of West-Ontario
[*] Class VII
Viruses: DNA Genomes with an RNA Replication Intermediate (Hepadnaviruses, Caulimoviruses)
[**] Class
VI Viruses: RNA Genomes with a DNA Replication Intermediate (Retroviruses)
Diagram illustrating HIV-CaMV replication
element interchangability
(see 'CaMV promoter, hepatitis B and HIV viruses, and cancer' below for
commentary)
"Most of the retroviruses we currently know (many!) infect vertebrates, but as a group, they have been identified in virtually all organisms including invertebrates - evolutionarily successful design!"
University of Leicester, Department of Microbiology & Immunology, Course BS335:
Virology - see page on retroviruses
The use of the
Cauliflower Mosaic viral promoter (CaMV 35S) , Joseph Cummins
Professor
Cummins - more on viral recombination effects in transgenic plants
The Virus Hazard
John Innes Centre confirms CaMV promoter has recombination hotspot
"A recent study of transgenic rice carried out at the John Innes Institute supports previous evidence that there is a 'recombination hotspot' in the CaMV 35S promoter. A recombination hotspot is a site prone to recombination, ie, breaking and joining with other DNA. Furthermore, some of the recombination events are 'illegitimate' or nonhomologous, and do not require substantial similarity in nucleic acid base sequence.
The results show that the CaMV promoter is very likely to recombine with other DNA in the host genome, including dormant viral DNA, as well as with other viruses in the host cell. Transgenic lines containing CaMV promoters, which includes practically all that have been released, are therefore prone to instability due to rearrangements, and also have the potential to create new viruses or other invasive genetic elements.
Such elements cannot be contained or controlled once they have entered the wider environment. It is now indisputable that recombination events will take place at the CaMV promoter in the current generation of transgenic plants. The continued release of such transgenic plants is unwarranted especially in the light of the new findings."
Angela Ryan, Molecular Biologist, Institute of Science in Society and Biology Department, Open University, on work carried out by the John Innes Centre on the Cauliflower Mosaic Virus promoter used in most transgenic plants and published in The Plant Journal 17(6), pp 591-601 (March, 1999).
Viral danger
from GM crops confirmed - John Innes Study details
CaMV 35S promoter fragmentation hotspot
confirmed, and it is active in animals
AIDS virus also has recombination hotspot
- click here for full ISIS report
(extract below)
"Dr. Veljkovic's group, in collaboration with groups in UK, Italy and US, isolated HIV-1 viral sequences carrying the complete Chi recombination hotspot (GCTGGTGG) from the blood of three out of 11 AIDS patients. In one of the patients, recombination had occurred at this point with a gene from the bacterium, Haemophilus influenzae.
In fact, this is not the first time that recombination has been identified involving the AIDS virus. The authors suggest that such recombination may have been involved in a number of other cases reported in the literature.......
[Dr Veljkovic advises as follows:] 'I would like to point out another possible problem with CaMV promoter. We have demonstrated that Chi recombination hot spot is responsible for in vivo recombination between HIV and bacteria H. influenzae ... On the other hand, the prion gene contains repeats of this recombination promoter. It will be interesting to see consequences of possible interaction between these two recombination hot spots (Chi and CaMV) after massive substitution of the bone-meal by the GM cattle-food.'......... "
Click here for additional research from
the JIC on CaMV and other GM crops risks
Extract below taken from authors’ reply to critiques on "Cauliflower Mosaic Viral Promoter – A Recipe for Disaster?" by Hoe, M.W., Ryan, A.and Cummins, J. (1999). Microbial Ecology in Health and Disease
"....There is no evidence that CaMV promoter per se is active in animal cells. However, on account of the modularity of its structure, small motifs and elements within the CaMV promoter may be interchangeable with those of the animal viruses. HIV is a retrovirus that depends on integration into the host genome for replication, and its complementary DNA will therefore be present in the genome of humans infected with the virus. Hepatitis B virus, being a pararetrovirus, does not require integration for replication, but its genome has, nevertheless, been found in human chromosomes in liver tissue, and is associated with liver carcinoma (see ref. 19).
The initiation site for the +DNA strand synthesis in CaMV is a polypurine tract (PPT). It has recently been reported that the PPT from HIV-1 gives up to 50% of the efficiency for CAMV +DNA strand synthesis as CaMV’s own element (29). All eukaryotes and prokaryotes share the core TATA box promoter element. It is not inconceivable that the TATA box, as well as other elements and motifs within the CaMV promoter, when recombined with dormant animal viral promoters, may reactivate the virus, generate new viruses or give functional viral promoters that make cellular oncogenes over-express, resulting in cancer...."
Diagram illustrating HIV-CaMV replication element interchangability
'Evidence
(1999) that a plant virus switched hosts to infect a vertebrate and then recombined with a
vertebrate-infecting virus' - click here for paper abstract
(Proc Natl Acad Sci USA, 96(14): 8022-7 1999 Jul 6)
Explanitory extracts from paper :
'Sometimes viruses are transmitted to a host species that they have not previously infected or that they rarely infect. Several of these atypical interspecies transmission events (host-switching events) have led to disease outbreaks in this century (1-3).....similarities between the genomes of some plant-infecting, vertebrate-infecting, invertebrate-infecting, and microbe infecting viruses indicate that they have common ancestors (5), and suggest that at some point, ancestral viruses switched between these very different kinds of hosts. These more radical changes in host preference have led to the evolution of many new virus species. They are significant in terms of both disease and evolution; beyond that, little about them is known: neither the identity of the original hosts, nor the possibility of linkage between the change in host preference and a specific genetic change.
The history of viruses is further complicated by interspecies recombination. Distinct viruses have recombined with each other, producing viruses with new combinations of genes (6,7); viruses have also captured genes from their hosts (8,9).....
Interspecies recombination between viruses has been linked to disease outbreaks (10), and some analyses suggest that it may be linked to host-switching (7,11)....'
Risks associated with Genetically Modified (GM) Baculovirus Vectors to Control Insect Pests
Additional viral recombination risks in transgenic crops
In addition to the use of the CaMV promoter in the majority of transgenic plants, some GM plants are also modified with the intention that they are able to resist attack from viruses in the environment. A number of such crops are already being released in various parts of the globe.
However, the scientific literature reveals that such crops can also be susceptible to recombination and other events which may enable the creation of new viral diseases. The literature confirms that it may be very difficult to prevent such phenomena when GM crops of this type are released into the environment.
For more on this particular type of risk from transgenic crops see extracts from Hull R. (1998) Detection of risks associated with coat protein transgenics. In: Methods in Molecular Biology: Plant Virology Protocols: from Virus Isolation to Transgenic Resistance (Eds: Foster G.D., Taylor S.C.). New Jersey, Humana Press Inc. 81, 574-555. - click here.
The author of the paper is Professor Roger Hull, of the Department of Virus Research at the John Innes Centre, UK.
Additional risks associated with crops
incorporating viral transgenes
identified by Monsanto and USDA Scientists
Sleeping Viruses Lurk in Plant Genomes
( Taken from ISIS
NEWS (Issue #3) )
A new study provides evidence of repeated integration of pararetroviral-like sequences
into the genome of tobacco at a copy number of approx 10,000. Therefore, plant
pararetroviruses may integrate much more commonly into host chromosomes than has been
previously thought. Furthermore, the insertions are thought to have occurred by
illegitimate recombination. Plant viral sequences were
thought to integrate rarely, if at all, into host genomes and this new evidence calls for
a reconsideration of this view. This has considerable implications for
plant genome evolution as integrated pararetorviral DNA could act as an insertional
mutagen or contribute strong constitutive promoters to neighboring plant genes or could
accumulate to generate a new repetitive sequence family.
Reference; Jakowitsch J et al (1999) Integrated pararetroviral
sequences define a unique class of dispersed repetitive DNA in plants. PNAS Vol 96
No23 pp 13241-13246
Our [ISIS] comment: This paper provides evidence that dormant viral DNA may be much more widespread in plant genomes than previously thought. It highlights the need for more extensive research in this area and it also has a bearing on the ecological impact of GM plants - although the authors of this paper fail to mention this. The CaMV 35S promoter is a pararetroviral-derived sequence used in most transgenic constructs, where it is integrated at random into the host genome. Furthermore, it contains an independent recombination hotspot and may therefore recombine with dormant viral sequences, which are in some instances integrated at extraordinarily high copy numbers and are also highly recombinogenic - as shown by the amount of illegitimate recombination events detected in this study. This may result in the reactivation of dormant viral sequences, novel epigenetic effects and other genetic disruptions, all of which are potentially hazardous and unpredictable (see Viral Gene Switch – A Recipe for Disaster? This issue). (A.R.)
The CaMV promoter and 'gene silencing'
The CaMV promoter may also give rise to 'gene silencing' phenomena - where genes are unintentionally switched off. Below are some comments by scientists on the nature and implications of this for the behaviour of transgenic crops:
Extract from: Dale P.J., Al-Kaff N., Bavage A., Irwin J., Senior I. (1998) Transgene expression and stability in Brassica. ACTA Horticulturae 459, 167-171. John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK (For full paper - click here)
".....Recent research in our laboratory with Brassica napus plants containing the 35S promoter from the mosaic virus (CaMV) has shown that upon infection with the CaMV the driven transgene is silenced (Al-Kaff et al unpublished). Intensive research at present is directed towards understanding this silencing mechanism and its significance.
As the 35S promoter is widely used to regulate transgenics in brassicas, it is important that we strive to obtain a clear understanding of the mechanisms of this silencing and its significance. This is important for two reasons, for assessing the use of the 35S promoter in agriculture and also for assessing the significance of this effect for biosafety...."
Extract from: 'Genetic Engineering is not an extension of conventional plant breeding' Michael K. Hansen Ph.D, Consumer Policy Institute/Consumers Union, USA, January 2000 (For full paper - click here )
".......Virtually all crop plants derived via GE also contain a powerful promoter (a genetic regulatory or "on"-switch) from the Cauliflower mosaic virus (the CaMV 35S promoter), which in nature causes a disease in plants in the mustard family.....The CaMV 35S promoter is used precisely because it is such a powerful promoter, which leads to hyperexpression of the transgenes, having them be expressed at perhaps 2 to 3 orders of magnitude higher than of the organism's own genes. The CaMV 35S promoter effectively puts the transgene(s) outside of virtually any regulatory control by the host genome as the natural plant promoters for each gene allow....
Use of such strong promoters also raise safety concerns. Since the CaMV 35S is so strong, not only can it affect the introduced transgenes, it can also affect genes (either turn them "on" or turn them "off") thousands of base pairs upstream and downstream from the insertion site on a given chromosome and even affect behavior of genes on other chromosomes. Consequently, depending on the insertion site, a gene that codes for a toxin could be turned "on," leading to production of that toxin...."Plant DNA viruses and gene silencing - Plant Molecular Biology
New Ways to Silence Transgenes
( Taken from ISIS NEWS (Issue #3) )
Reference: Hamilton A.J, Baulcombe D.C (1999). Science
286, 950-952 also see “Silent Saboteurs” NewScientist, 6.11.99 p 25.
Summary by Institute of Science in Society: Researchers at the John Innes
Center in Norwich finds a new species of small antisense RNA molecule involved in post
transciptional gene silencing (PTGS) in plants. PTGS represents a natural antiviral
defense mechanism, which works against any invasive foreign genetic element that finds
expression. Transgenic RNA in GM plants are frequently targeted by PTGS mechanisms
and this strongly suggests that transgenes are perceived as viruses
by their host cell. This study has detected antisense RNA that is uniform in
length - approx. 25 nt (nucleotides) and complementary to the targeted mRNA. It has
been term ‘spoiler RNA’ for it forms a duplex with the target RNA and promotes
degradation as well as interfering with translation. It has been shown to accumulate
in cells, either when transgene transcription or RNA virus replication is taking
place. The size of the spoiler RNA is also significant - it is small enough to
move through the plasmodesmata (pores between cells) and has been shown to spread into
nearby cells and activate PTGS elsewhere in the plant. The precise role of the 25nt
RNAs in PTGS is yet to be elucidated, but it is suggested these are components of a
systemic signal and specificity determinant in PTGS.
Comment by Institute of Science in Society: This
new discovery shows that GM plants respond to transgenes in the same way they do
viruses. Transgenic RNA transcripts are produced at a very high copy number
in GM plants and are under the control of powerful promoters like the CaMV 35S
promoter. Viral infection causes metabolic stress in cells and can lead to
PTGS. The same can now be said of transgenic constructs in GM plants.
Furthermore, the size and migratory nature of the RNAs reveal how small neucleotides have
very important biological functions in cells. This calls for regulation of all naked
or free nucleic acids used in genetic engineering biotechnology, a point made forefully by
Traavik (1999). Too Early May be Too Late: Ecological Risks of Naked
DNA, Report to Directorate of Nature Management, Trondheim, Norway.
Naked DNA in GM crops
(Commentary by Professor Joe Cummins - extracts)
"Naked DNA is DNA that has been stripped of protein in the laboratory or exposed to natural detergents or phenols in the environment. For example, viruses stripped of protein may be taken up by cells and infect animals or people that are not a part of the normal host range of the virus....
Naked DNA may be produced during exposure to GM crops during ingestion of the crop as food, breathing pollen , exposure to cut or wounded plant parts or to exposure to decaying plant material in the field or waste. Once plant DNA or plant virus DNA is taken up by human cells the protein coding sequences may be spliced to the chromosomal DNA downstream of a promoter or the LTR sequence of a retrotransposon and be expressed under control of the human regulatory sequences.....
In the past it has been assumed that naked DNA is rapidly destroyed on exposure to the animal digestive systems or microbes in water or soil. As experimentalists became daring it was found that DNA may be taken up by cells and express its genes ....."
Full commentary and references by Professor Cummins - click here
Genetic Engineering Superviruses - ISIS report
Viral danger from GM crops confirmed - John Innes Study details
Why genetic engineering is
not science based
Fundamental scientific conceptual errors in the development of recombinant DNA
technology
What leading scientist have said about the dangers of
genetically modified crops and foods
