The current issue of the Genetic Engineering Newsletter
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Genetic Engineering Newsletter - Special Issue 6
February 2001
supported by
Gerling-Foundation, Triodos-Stichting, Mahle-Foundation and Zukunftsstiftung
Landwirtschaft
Pleiotropic and Position Effects - unintended effects in genetic engineering
CONTENTS
Preface
Definitions
Pleiotropic effect
Position effect
Examples with microorganisms
E. coli and indigo blue - happy end after all?
Yeast - more alcohol, more dangerous substances?
Examples with plants
Arabidopsis thaliana - examples for all kind of effects
Petunia - salmon colored in the greenhouse, mottled in the field
Herbicide-tolerant soybeans - heat sensitive
Roundup Ready cotton - stress sensitive
Transgenic rapeseed - inducible seed dormancy
Transgenic potatoes - Change in morphologic and phenotypic characteristics
Transgenic trees - early blossom phenomen
Examples with animals
Transgenic fish - change in hormone levels
Risk aspects
References
Preface
Even though these days, DNA-sequences are relatively easy to decipher, the
knowledge and understanding of complex higher contexts and interactions within
the genome is rather small. One and the same gene may have different characteristics
and effects (so called pleiotropic effects).
Furthermore, depending on the spot of insertion, one gene may have different
meanings due to the influence of the surrounding genes (so called position effects).
That means, in different organisms or in different contexts, the same genes
may lead to different characteristics. Unintended, additional side effects
are subject of this Special Issue. However, pleiotropic and position effects
are not systematically described in scientific literature. For example, transgenic
plants having these effects frequently produce worse agronomic results than
the not genetically modified parent-lines and therefore are sorted out in the
developing process in the laboratory. For this reason, only several examples
are listed in this issue.
Definitions
Pleiotropic effect
Pleiotropy means that one gene may be responsible for the development of several
features and characteristics. Pleiotropic effect describes an often
unforeseen change of several characteristics in transgene and non-transgene
organisms, when only one characteristic was about to be changed. Therefore,
pleiotropic effects may cause various phenomena and processes in organisms.
These are mainly changes in cell metabolism, which could lead to phenotypic
(Phenotype: the total characteristics displayed by an organism under a particular
set of environmental factors, regardless of the actual genotype of the organism.
Results from the interaction between the genotype and the environment [On-line
Medical Dictionary, 12.02.01, http://www.graylab.ac.uk/cgi-bin/omd?query=phenotye]
) changes in plants (LIPS, 1998).
In some cases, the phenomen of gene silencing is defined as a pleiotropic
effect, but it will not be highlighted in this issue. The reason for gene silencing
is a reduced amount of mRNA (mRNA is a copy of the gene (or transgene), which
is needed to transfer the genetic information to where the encoded gene product
is finally produced ) of the specific transgen, which means that the transgen
exists, but its expression is extremely reduced or totally absent (MITTELSTEN-SCHEID,
1995).
Position effect
This term describes the influence of the gene's position to its activity. That
means, these are effects based on the fact, that different intensity and effects
of one gene to other genes are dependent from its position in the DNA.
Scientific literature more often pays attention to position effects than to
pleiotropic effects, since according to actual knowledge, the position of a
gene has influence on the extent and stability of gene expression. If alterations
in metabolism or phenotypic changes are detected, usually it cannot be traced
if the effect is pleiotropic or due to the gene's position or even a combination
of both.
Examples with microorganisms
Environmental effects of genetically modified microorganisms are rarely tested, though they are in daily use from laboratory to industrial plants. Therefore, data for pleiotropic and position effects are rare. E. coli and indigo blue - happy end after all? One of the earliest examples for pleiotropic effects of transgens in microorganisms has been described for genetically modified Escherichia coli. In 1983, scientists already published unexpected results with transgenic E. coli bacteria. Genes which should enable the breakdown of naphthalene to salicylic acid had been inserted into the bacteria. Additionally to this breakdown, the bacteria afterwards produced indigo blue. The integration of the new enzymes enabled the bacteria to carry out the transformation of the existing indol to indigo blue. In the meantime, this unexpected discovery is used for industrial production (AMATO, 1991).
Yeast - more alcohol, more dangerous substances?
The production of certain enzymes for an improved alcohol fermentation in transgenic yeast lead to an increased building of methylglyoxal, a mutagenic compound, which in non-transgenic yeast is only detectable in traces, if any at all. Obviously, aside from the normal glycolytic pathway, through genetic engineering another metabolic pathway has been induced by the high enzyme level (IMOSE AND MURATA, 1995).
Examples with plants
In the area of transgenic plants, pleiotropic effects mainly have been tested and detected with herbicide-tolerant plants. In the context of pleiotropic changes, CALAGENE Inc. (1990) stated that one third of all transgenic plant lines show pleiotropic effects not connected to the nature of the inserted gene or the gene product. Despite the high occurrence of the phenomenon, there are almost no scientific papers since those plants are sorted out within the developing process of transgenic lines.
Arabidopsis thaliana
Arabidopsis thaliana is regarded as THE model plant for genetic engineers. This plant also provides examples for unwanted effects caused by genetic engineering interventions into the genome. BERGELSON ET AL. (1996) tested effects of a herbicide resistance gene on the fitness of the plants. Seed production was one of the indicators for fitness. Transgenic Arabidopsis-plants were compared to a non-transgenic line, which had adopted the herbicide resistance by mutation, and to the non-transgenic parent-line. The examination showed that the transgenic plants had a reduced fitness which undoubtedly was caused by pleiotropic effects of the herbicide resistance gene. Usually, Arabidopsis is strictly self-fertile. Some genetically modified, herbicide-resistant Arabidopsis plants showed an increased tendency for cross-pollination. This effect turned out to be a combination of pleiotropic and position effects. Especially in cruciferous plants, like Arabidopsis is, cross-pollination is directly connected to a higher risk of out-crossing of (transgenic) characteristics.
Petunia - salmon colored in the greenhouse, mottled in the field
First field trials of GM plants in Germany became famous especially because
of being a prominent example for position and pleiotropic effects. Transgenic
petunia were supposed to show salmon-colored petals after the insertion of a
certain maize gene. In greenhouse experiments, the petunia reacted as expected,
but when released in fields, they showed several unwanted effects. Mainly flowers
were white or mottled. Furthermore the plants had more leaves and shoots while
their fertility was reduced. To pathogenic fungi, they resisted better than
their non-transgenic parent-lines. Follow-up experiments identified those effects
as gene silencing, which additionally was dependent on environmental factors
(MEYER ET AL., 1992).
Herbicide-tolerant soybeans - heat sensitiveness
A 20% increase in lignin (Lignin is the woody compound of plant cells giving
them stability. But its storage also reduces the supply with water and nutrients
and reduces the elasticity) -production in Roundup Ready soybeans is suspected
being caused by the newly inserted enzyme which has unexpected effects on the
lignin metabolism and therefore causes a lignin-overproduction in the plants
(GERTZ ET AL., 1999, COGHLAN, 1999). Under stress conditions, this effect is
negative for transgenic soy-plants. An induced water shortage reduced the fresh
weight of the transgenic plants by 48%, whereas the non-GM plants only had 24%
reduction of fresh weight. Also heat stress lead to reduced yields in transgenic
soy (VENCILL, 1999).
Furthermore, a change in plant hormones was detected in the transgenic soy,
with a decrease up to 14 % (LAPPE ET AL., 1999). Roundup Ready cotton - stress
sensitiveness In 1997, cotton which was genetically engineered being resistant
to the herbicide Roundup and which was commercially grown in the US-State Mississippi
showed capsule-deformation and -dropping. More than 200 farmers therefore had
to bear an enormous economic backlash (HAGEDORN, 1997). Though no thorough analysis
to understand the phenomena has been done, one could imagine that the herbicide-resistant
cotton is similar stress sensitive as Roundup Ready soy due to pleiotropic effects.
Transgenic rapeseed - inducible seed dormancy Experiments with transgenic rapeseed
lines with high-stearate and high-laurate content showed pleiotropic effects
important to risk assessment: Both transgenic lines showed a higher rate of
inducible seed dormancy. This could lead to an increased risk of building a
seed bank and thereby to the establishment of wild transgenic populations (LINDER,
1998).
Transgenic potatoes - change in morphologic and phenotypic characteristics
Changes in the basic plant metabolism through genetic engineering could cause pleiotropic effects, detectable in changes in plant tissue compounds or changed morphologic or phenotypic characteristics. For example, transgenic, fructane-building potatoes which have been examined by BECKER ET AL. (2000) were observed having a different flowering behavior and different shoot length. Furthermore, a significant reduced yield was observed. Earlier trials with transgenic potatoes which had been modified in phosphate- and carbohydrate-metabolism produced similar results (BECKER ET AL., 1998). Transgenic trees - early blossom phenomenon
Transgenic trees tested in field trials very often show pleiotropic and/or
position effects. Greenhouse experiments already revealed that poplars, genetically
engineered with a certain promoter (Promoter: A region of DNA, involved in the
regulation of the expression of a gene.) could be earlier in blossom than non-transgenic
control groups (FLADUNG ET AL., 1999). A significant higher level of phytohormones
which has been observed with transgenic poplars is suspected causing the phenomena
(FLADUNG ET AL., 1997). Phytohormone-levels are generally connected to blossom
behavior of plants. Some transgenic poplars tested in a field trial which started
in 1996, already showed female flower-buds after 3 years, though naturally
poplars only start being in blossom after 8 years.
If male flower-buds would be built, the risk for spread of transgenic genes
is immense due to hybridization possibilities of poplars. Particularly the genera
populus, eucalyptus and pinus - genera where in genetic engineering is very
common - are known for their high hybridizat ion potential. In northern, temperate
regions poplars, for example, have up to 30 species
with several hybrids (DIAFOZIO ET AL., 1999).
Examples with animals
Transgenic fish - change in hormone levels
Several genetically engineered fish show severe pleiotropic effects. Very often genes coding for growth hormones are inserted into fish, leading to a disturbance of the balance of growth hormones. An immense growth increase in transgenic salmon, for example, was followed by severe deformation of the head and other parts of the body and furthermore by a changed fat deposition (DUNHAM, 1999).
A summary of body deformations of transgenic fish given by PANDIAN ET AL. (1999) list the following changes already appeared: tumors, changed shape of fins and vertebras, deformation of the head, abnormal growth of gills, absent body segments, stunted shape of neck and tail. Aside from pleiotropic effects, transgenic fish are known to have problems in stabile gene expression. Additionally, mosaicism - that is, within a modified individual there are both cells with and without the transgene - is a regularly occurring phenomenon in transgenic fish. The number of modified sequences varies from cell to cell, from organ to organ, and from individual to individual. Especially for genetically modified growth hormones, there are several examples for this phenomenon, e.g. in catfish, zebrafish and carp.
Risk aspects
Pleiotropic and position effects are risks arising from the methods of genetic
engineering. They occur relatively often, though there is no special observance
for them. The description of those effects is dependent on the intensity the
transgenic organisms were examined. Such described effects range from changes
in agronomic characteristics to permanent variations of the gene
expression levels. If such effects occur during the development of transgenic
organisms, those usually are sorted out. However, pleiotropic and position effects
pose a risk if they cause new
possibilities for the spread of transgenes, or are responsible for the production
of toxic or allergic compounds (LIPS, 1998). Risk which are discussed in the
context of pleiotropic and position effects are:
- the insertion of foreign proteins/enzymes could lead to unforeseeable changes in the metabolism and thereby lead to new, potentially toxic or allergic gene products;
- the insertion of foreign DNA-sequences may influence or even destroy genes at the point of integration and thereby disturb the genome context or may lead to toxic or allergic metabolites;
- organisms are able to influence or even stop the expression of inserted foreign genes and therefor a continuos gene expression cannot to be guaranteed (LIPS, 1998).
- Effects on secondary plant components.
References
AMATO G (1991) Species hybridization and protection of endangered animals, Science,
253: 250.
BECKER R, AUGUSTIN J, BEHRENDT U, GRANSEE A, HEDTKE C, LUETTSCHWANGER D, MUELLER
M, ULRICH A (2000) OEkologische Begleitforschung zum Anbau von transgenen Kartoffeln
mit Veraenderung im Grundstoffwechsel. Landesumweltamt Brandenburg, Muencheberg.
BECKER R, MARTY B, ULRICH A (1998) Experimentelle Verifizierung von Veraenderungen
risikorelevanter oekologischer Parameter bei transgenen Kartoffeln mit Veraenderungen
im Phosphat- und Kohlenhydratmetabolismus. Landesumweltamt Brandenburg, Muencheberg.
BERGELSON J, PURRINGTON CB, PALM CJ, LOPEZ-GUTIERREZ JC (1996) Costs of resistance:
a test using transgenic Arabidopsis thaliana. Proceedings of the Royal Society
of London, B, 263: 1659-1663.
CALAGENE INCORPORATED (1990) KanR Gene: Saftey and Use in the Production of
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DIFAZIO SP, LEONARDI S, CHENG S, STRAUSS SH (1999) Gene flow and agriculture;
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DUNHAM RA (1999) Utilization of transgenic fish in developing countries: Potential
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