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 Genetically Engineered Plants. Request for Advisory. Calagene Incorporated Vol 1 of 2: 233, 1920 Fith Street, Davis California 95616 zitiert aus:
LIPS J (1998) Pleiotrope Effekte und genetische Stabilitaet transgener Pflanzen. In: SCHUETTE G, HEIDENREICH B, BEUSMANN V (1998) Nutzung der Gentechnik im Agrarsektor der USA - Die Diskussion von Versuchsergebnissen und Szenarien zur Biosicherheit, UBA-Texte 47/98: 121-156.
COGHLAN A (1999) Splitting Headache, New Scientist, 20. Nov. 1999.
DIFAZIO SP, LEONARDI S, CHENG S, STRAUSS SH (1999) Gene flow and agriculture; relevance for transgenic crops. Proceedings of a symposium held at Keele, UK on 12-14 April 1999, BCPC Symposium Proceedings No 72:171-176.
DUNHAM RA (1999) Utilization of transgenic fish in developing countries: Potential benefits and risks. Journal of the World Aquatic Society, 30(1).
FLADUNG M, GROSSMANN K, AHUJA MR (1997) Alterations in hormonal and developmental characteristics in transgenic Populus. Journal of Plant Physiology, Germany, 150: 420-427.
FLADUNG M, NOWITZKI O, EBBINGHAUS D, SCHELLHORN A, BENTIEN G, AHUKA MR, MUHS HJ (1999) Field relase of ROLC-transgenic Aspen-Populus. Online:
http://users.ox.ac.uk/~dops0022/conference/forest_biotech99_home.html Poster 47, 3.12.1999.
GERTZ JM (JR.), VENCILL WK, HILL NS (1999) Tolerance of transgenic soybean
(Glycine max) to heat stress. British Crop Protection Conference, 15-18 November 1999 - Weeds, Proceedings of an International Conference, Brighton, 3: 835-840.
HAGEDORN C (1997) Boll drop problems in roundup-resistant cotton. Crop and Soil Environmental News, 12/1997.
INOSE T, MURATA K (1995) Enhanced accumulation of toxic compund in yeast cells having high glycolytic activity: a case study on the safety of genetically engineered yeast. International Journal of Food Science and Technology 30: 141-146.
LAPPE MA, BAILEY EB, CHILDRESS C, SETCHELL KDR (1999) Alterations in Clinically important phytoestrogens in genetically modified, herbicide-tolerant soybeans. Journal of Medicinal Food, 1(4).
LINDER CR (1998) Potential persistance of transgenes: Seed performance of transgenic canola and wild x canola hybrids. Ecological Applications 8: 1180-1195.
LIPS J (1998) Pleiotrope Effekte und genetische Stabilitaet transgener Pflanzen. In: Schuette G, Heidenreich B, Beusmann V (1998) Nutzung der Gentechnik im Agrarsektor der USA - Die Diskussion von Versuchsergebnissen und Szenarien zur Biosicherheit. UBA-Texte 47/98: 121-156.
MEYER P, LINN F, HEIDANN I, MEYER H, NIEDENHOF I, SAEDLER H (1992) Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype. Mol Gen Genet 231: 345-352.
MITTELSTEN-SCHEID O (1995) Transgene Inactivation in Arabidopsis thaliana. In: MEYER P (Ed.) Gene Silencing in Higher Plants and Related Phenomena in Other Eukaryotes: 29-43, Springer Verlag Berlin.
PANDIAN TJ, VENUGOPAL T, KOTEESWARAN R (1999) Problems and prospects of
hormone, chromosome and gene manipulation in fish. Current Science 76: 369-386.
VENCILL WK (1999) Increased susceptibility of glyphosate-resistant soybean to stress (abstract). In: British Crop protection Council 8eds.) The 1999 Brighton Conference - Weeds.


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