GMW: Scrambling and Gambling with the Genome

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GMW: Scrambling and Gambling with the Genome

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Tue, 2 Aug 2005 20:26:49 +0100

 

 

 

 

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Scrambling and Gambling with the Genome

By Jeffrey M. Smith, author of Seeds of Deception

Spilling the Beans, July 2005

 

" With genetic engineering, transferring genes from one species' DNA to

another is just like taking a page out of one book and putting it

between the pages of another book. " This popular analogy is used often by

advocates of genetically modified (GM) food. The words on the page are

made up of the four letters, or molecules, of the genetic code, which

line up in " base pairs " along the DNA. The inserted page represents a

gene, whose code produces one or more proteins. The book is made up of

chapters, which represent chromosomes­large sections of DNA.

 

The analogy makes the process of genetic engineering appear to be as

simple and precise as inserting a new page. A groundbreaking report,

however, shreds the book analogy. Genome Scrambling – Myth or Reality?,

written by three scientists at the UK-based Econexus, reveals that the

process of genetic engineering results in widespread mutations­ within

the

inserted gene, near its insertion, and in hundreds or thousands of

locations throughout the genome­ and that these are overlooked by many

scientists and regulators. [1]

 

The report is an extensive review of research that overturns the

central arguments by biotech advocates ­that the technology is precise,

predictable, and safe, and that current studies are adequate. On the

contrary, it demonstrates that GM crops represent a significant gamble to

public health and the environment (see http://www.econexus.info/ ).

 

Gene Insertion Methods Create Mutations, Fragments, and Multiple Copies

 

There are two popular methods for creating GM crops; both create

mutations. The first method uses Agrobacterium­bacteria that contain

circular

pieces of DNA called plasmids. One section of this plasmid is designed

to create tumors. Under normal conditions, Agrobacterium infects a

plant by inserting that tumor-creating portion into the plant's DNA.

Genetic engineers, however, replace the tumor-creating section of the

plasmid

with one or more genes. They then use the altered Agrobacterium to

infect a plant's DNA with those foreign genes.

 

The second method of gene insertion uses a gene gun. Scientists coat

thousands of particles of tungsten or gold with gene sequences and then

shoot these into thousands of plant cells. Years ago, the sequences that

were shot into cells usually included both the genes that were intended

for transfer (gene cassette) as well as extraneous DNA from the plasmid

used in the creation and propagation of the cassettes in bacteria. This

is true for most GM foods currently on the market. These days, many

scientists take the added step of eliminating the extraneous, mostly

bacterial DNA and coat the particles just with the cassette.

 

With both methods of gene insertion, scientists speculate that the

process triggers a wound response in the plant cell, which helps its DNA

integrate the foreign gene. With the gene gun technique, only a few cells

out of thousands incorporate the foreign gene.

 

According to the book analogy, a single, intact, foreign page (gene) is

inserted. That's the intention. In reality, most transformed DNA end up

with multiple copies of the foreign gene, incomplete genes and/or gene

fragments. Sections of the inserted genes are commonly changed,

rearranged, or deleted during the insertion process. In addition,

extraneous

pieces of plasmid DNA sometimes end up interspersed within and around

the inserted gene or scattered throughout the genome.

 

Mutations Near the Site of Insertion

In addition to the changes made in the material that is inserted, the

sections of the plant's DNA near the insertion site are almost always

messed up in some way. This effect, called insertional mutagenesis or

insertion mutation, has been known for years, but it wasn't until 2003

that a large-scale systematic analysis was conducted. Researchers looked

at insertions into 112 Arabidopsis thaliana plants­a species used often

in plant research. [2] Although the study may not accurately reflect

what happens in edible crop plants, it is the only large study at this

point.

 

Plants were selected that had single copies of the foreign gene, which

were inserted using Agrobacterium. Of the 112 plants, 80 (71%) had

small mutations near the insertion site. These included deletions of

1-100

base pairs and/or insertions of 1-100 extraneous base pairs. The

inserted sequences came from the foreign gene, extraneous parts of the

plasmid, or other parts of the plant's DNA.

 

The remaining 32 plants (29%) had large scale insertions,

rearrangements, duplications and/or deletions. In two plants, parts of

whole

chromosomes had broken off and translocated into another section of

the DNA.

 

Another study using the same plant species also found that a section of

DNA at least 40,000 base pairs long had translocated from one

chromosome to another. In fact, that long section had duplicated

itself, since

it was also found intact in its original position. [3] A third study

identified a deletion of 75,800 base pairs, which probably contained 13

genes. [4]

 

The above studies used the Agrobacterium insertion method. There have

been astoundingly few studies analyzing insertion mutations resulting

from the gene gun method, but the research that has been conducted

consistently demonstrate large scale disruptions of the DNA. According

to the

Econexus report, " The vast majority of the insertion events created via

particle bombardment [gene gun] are extremely complex, with multiple

copies of transgenic DNA inserted at a single insertion-site. " 1 They

contain large amounts of extraneous DNA, including multiple fragments of

the foreign gene and/or small or large fragments of plant DNA

interspersed with the inserted genes. In one study, scientists found

155 separate

breaks indicating recombinations of the inserted genetic material.

[5]According to the Econexus report, in the rare cases where only a

single

copy of the foreign gene is inserted, they " turn out to contain

fragments of superfluous DNA and/or they appear to be associated with

large

deletions and/or rearrangements of the target plant DNA. " 1

 

One study on gene gun insertion revealed that DNA of an oat plant

contained the full sequence of the foreign gene plasmid, a small

stretch in

which oat DNA was mixed up with foreign plasmid DNA, a partial copy of

the plasmid, and another section with oat and plasmid sequences

scrambled together. [6] Analysis also indicated that the plant's DNA

on either

side of the insertion contained rearrangements or deletions. There were

also two other insertions elsewhere in the DNA. One included a

rearranged section of the plasmid (296 base pairs), scrambled plant

DNA on

either side, and the deletion of 845 base pairs. The study employed DNA

sequence analysis, the most thorough method for evaluating insertion

mutations. In practice, it is rarely used. Instead, genetic engineers

traditionally rely on the less precise Southern blot test, which picks up

only major changes in DNA sequence. When this test had been applied to

the

oat DNA above, it indicated the presence of only a single intact

inserted gene. It failed to identify the other two insertions and all

of the

mutations and fragments. This means that on the whole, biologists who

create GM plants have no idea of the extent to which their creations may

produce unintended side effects do to scrambled DNA.

 

Location, Location, Location

 

Neither gene insertion method can " aim " the foreign gene into a

particular location in the DNA. Furthermore, scientists rarely conduct

experiments to find out where exactly the inserted genes end up. But

in the

real estate of the DNA, location is vital. The functioning of the foreign

gene can change dramatically, depending on where in the genome it is

located. The side-effects of gene insertion can be significantly

influenced by location as well.

 

Even though only an estimated 1-10% of plant DNA constitutes the genes,

Agrobacterium insertions end up inside functioning gene sequences

between 35%-58% of the time. (The percentage for gene guns is unknown.)

Genes are also inserted in other areas that influence gene expression. In

either case, insertions can significantly disrupt the normal functioning

of the plant's genes.

 

(One reason why insertions end up inside genes so often is that in

order for the foreign genes to function, they need to be located

within the

regions of the host DNA that are " active, " that allow gene expression.

To figure out which inserted genes end up in these portions of the DNA,

scientists typically add an antibiotic resistant marker (ARM) gene to

the genetic cassette. After insertion, they apply antibiotics to all the

cells, killing those that don't have a functioning ARM gene in their

DNA. Since the active region of the DNA is also where the plant's

functioning genes are located, those that survive this selection

process are

more likely to have foreign genes lodged inside the host genes.)

 

Mutations Throughout the DNA

 

Once genes are inserted into a plant cell's DNA, scientists typically

grow the cell into a fully functioning plant using a method called

tissue culture. Unfortunately, this artificial method of plant

propagation

results in widespread mutations throughout the genome. In fact, tissue

culture is sometimes used specifically to create mutations in plant DNA.

These mutations can influence the crops' height, resistance to disease,

oil content, number of seeds, and many other traits. [7][8]

 

Genetically modified cells that undergo tissue culture can have even

more mutations throughout the genome than cultured non-GM cells. It is

unclear why gene insertion has this effect, but scientists speculate that

it may, in part, come from unsuccessful insertions or insertions of

small fragments.

 

Taken together, the process of gene insertion combined with tissue

culture typically results in hundreds or thousands of mutations,

including

small deletions, substitutions, or insertions in the genetic code. The

changes are vast. Two studies suggested that 2-4% of the genome of a GM

plant was different than non-GM controls. [9][10] Furthermore,

estimates are based on detection methods that miss many mutations such

as short

deletions and insertions and most base pair substitutions. Thus, the

actual degree of gene disruption is probably greater.

 

These genome-wide mutations are found in every GM plant analyzed.

Astoundingly, these types of mutations are not evaluated in commercially

released GM food crops.

 

If the original GM plant is crossed (mated) with other lines over and

over, many of these small, genome-wide mutations will get corrected. It

is unknown, however, how many mutations still persist in food crops.

Furthermore, the propagation of certain species, such as the GM potato

that was on the market years ago, probably did not undergo any

outcrossing, and it is likely to contain all of the mutations created

during

insertion and tissue culture.

 

Mutations Can Have Serious Consequences

 

Mutations and extraneous insertions carry risk. They can permanently

turn genes on or off, alter their function, and/or change the structure

or function of the protein that they create. A single mutation can

influence many genes simultaneously. Thus, the insertion process might

cause

the over production of toxins, allergens, carcinogens, or

anti-nutrients, reduce the nutritional quality of the crop, or change

the way that

the plant interacts with its environment. And because of our limited

understanding of the DNA, even if we knew which parts of it were

disrupted, we wouldn't necessarily know the consequences.

 

In addition, the insertion of bacterial plasmid DNA into plant DNA

creates another serious risk. Similarities in the genetic sequence

between

the plasmid and the DNA of bacteria found in the gut of humans or

animals or in the soil might significantly increase the likelihood of

horizontal gene transfer. This means that genes from the plant may

transfer

into the DNA of the soil or gut bacteria. The only human feeding study

on genetic engineering confirmed that the genes inserted into GM

soybeans do transfer into the bacteria inside human intestines.

 

Advocates of biotechnology often defend the safety of their products by

claming that modern methods of plant breeding other than genetic

engineering are used on a wide scale, have a history of safe use and

create

comparable mutations. The Econexus report reveals that everything about

this argument is pure speculation and is not supported by scientific

literature. There is no evidence that these modern methods are used

widely, are consistently safe, or create mutations of the same kind or

frequency as genetic engineering.

 

In reality, many biotech scientists are unaware of the massive quantity

of mutations that are generated by the GM transformation process (gene

insertion and tissue culture). In fact, the regulatory agencies that

approve GM foods operate as if the insertion process has no impact on

safety. [11][12] They do not require extensive evaluation of the

mutations

and therefore the extent of these in approved GM food crops has not

been identified. The few studies that have been conducted revealed many

significant problems. GM varieties contain truncated or multiple

fragments of the inserted gene and extraneous or scrambled DNA. One GM

corn

variety contained a fragment from a gene that was supposed to be inserted

into a different GM variety. The protein produced by the foreign genes

can also be truncated, altered, or fragmented. And many significant

differences between GM and non-GM crops have been observed, which may

result from the insertion process. An approved GM squash, for example,

contains 68 times less beta-carotene and four times more sodium than

non-GM

squash. GM soybeans have much higher levels of a potential allergen and

anti-nutrient. But GM crops are tested for only a handful of nutrients

or known toxins, and therefore the true impact of gene mutations is not

known. Furthermore, GM plants are grown in vast amounts. Undetected

alterations may result in harm to the environment or human health on an

unprecedented scale. With so little known about the impact of gene

insertion, and with so much at risk, applying genetic engineering to

food and

crops is a huge gamble.

 

Revised Book Analogy

 

With genome scrambling in mind, let's revise the book analogy as

follows:

 

The DNA is like a large book with the letters consisting of the four

molecules that make up the genetic code. Located throughout the book are

special one- to two-page passages, called genes, which describe

characters called proteins (including enzymes). The book is divided into

chapters called chromosomes.

 

When a single foreign page (gene) is inserted through the process

called genetic engineering, the book goes through a profound

transformation.

There are typos throughout, in hundreds or thousands of places. Letters

are switched here and there; words and sentences are scrambled,

deleted, repeated or reversed. Long and short passages from one part

of the

book may be relocated or repeated elsewhere, and bits of text from

entirely different books show up from time to time. As you get close

to the

inserted page, things get really strange. The story becomes

indecipherable. The text includes random letters and sections of

inserted foreign

text, and several pages are missing. The inserted page may actually be

multiple identical pages, partial pages, or small bits of text, sections

that are misspelled, deleted, inverted, and scrambled. As a result of

changes in the story line throughout the book, several characters

(proteins) act differently, sometimes switching roles from heroes to

villains, or vice versa. It all makes you wonder about the comment

made by the

biotech advocate as he handed you the volume, " It's just the same old

book, only with a single page added. "

 

Spilling the Beans is a monthly column available at

<http://www.responsibletechnology.org/>. Publishers and webmasters may

offer this article or monthly series to your readers at no charge, by

emailing <column. Individuals may read

the column each month by subscribing to a free newsletter at

<http://www.responsibletechnology.org/>.

 

[1] Allison Wilson, PhD, Jonathan Latham, PhD, and Ricarda

Steinbrecher, PhD " Genome Scrambling -Myth or Reality?

Transformation-Induced

Mutations in Transgenic Crop Plants Technical Report - October 2004,

<http://www.econexus.info/>www.econexus.info. The references below were

cited in this report.

 

[2] Forsbach A, Shubert D, Lechtenberg B, Gils M, Schmidt R (2003) A

comprehensive characterisation of single-copy T-DNA insertions in the

Arabidopsis thaliana genome. Plant Mol Biol 52: 161-176.

 

[3] Tax FE, Vernon DM (2001) T-DNA-associated

duplication/translocations in Arabidopsis. Implications for mutant

analysis and functional

genomics. Plant Physiol 126: 1527-1538.

 

[4] Kaya H, Sato S, Tabata S, Kobayashi Y, Iwabuchi M, Araki T (2000)

hosoba toge toge, a syndrome caused by a large chromosomal deletion

associated with a T-DNA insertion in Arabidopsis. Plant Cell Physiol

41(9):

1055-1066.

 

[5] Svitashev SK, Pawlowski WP, Makarevitch I, Plank DW, Somers DA

(2002) Complex transgene locus structures implicate multiple

mechanisms for

plant transgene rearrangement. Plant J 32: 433-445.

 

[6] Makarevitch I, Svitashev SK, Somers DA (2003) Complete sequence

analysis of transgene loci from plants transformed via microprojectile

bombardment. Plant Mol Biol 52: 421-432.

 

[7] Dennis ES, Brettell RIS, Peacock WJ (1987) A tissue culture induced

Adh2 null mutant of maize results from a single base change. Mol Gen

Genet 210: 181-183.

 

[8] Brettell RIS, Dennis ES, Scowcroft WR, Peacock WJ (1986) Molecular

analysis of a somaclonal mutant of maize alcohol dehydrogenase. Mol Gen

Genet 202:235-239.

 

[9] Bao PH, Granata S, Castiglione S, Wang G, Giordani C,Cuzzoni E,

Damiani G, Bandi C, Datta SK, Datta K, Potrykus I, Callegarin A, Sala F

(1996) Evidence for genomic changes in transgenic rice (Oryza sativa L.)

recovered from protoplasts. Transgen Res 5: 97-103.

 

[10] Labra M, Savini C, Bracale M, Pelucchi N, Colombo L, Bardini M,

Sala F (2001) Genomic changes in transgenic rice (Oryza sativa L.) plants

produced by infecting calli with Agrobacterium tumefaciens. Plant Cell

Rep 20: 325-330.

 

[11] NRC/IOM: Committee on Identifying and Assessing Unintended Effects

of Genetically Engineered Foods on Human Health (2004) Safety of

Genetically Engineered Foods: Approaches to assessing unintended health

effects. The National Academies Press, Washington, DC.

 

[12] Kessler DA, Taylor MR, Maryanski JH, Flamm EL, Kahl LS (1992) The

safety of foods developed by biotechnology. Science 256: 1747-1832.

 

© Copyright 2005 by Jeffrey M. Smith. Permission is granted to

reproduce this in whole or in part.

 

 

 

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