Release of genetically engineered micro-organisms into the environment

Summary Genetically engineered micro-organisms (GEM) at present are the subject of much public attention. They are being considered for biological control, frost protection of plants, and other applications. There is a need to test such organisms before release to the environment. Examples of GEM includePseudomonas fluorescens into which have been cloned -toxin genes ofBacillus thuringiensis, ice-minus bacteria, and other organisms modified by addition, rearrangement and/or deletion of genetic material. Prior to release, the survival, fate, and effects of GEM in the environment must be established. Because organisms, once released, cannot be recalled or always controlled, it is imperative that a full understanding of the risks be known. Predictive ecology must include the new sub-discipline of molecular microbial ecology, if the need for information prior to release of GEM is to be met. One of the most important aspects of deliberate release which must be considered is the ability to detect and monitor GEM in the environment. It has been discovered that micro-organisms can undergo dormancy, i.e. enter a viable but non-recoverable stage in the natural environment. New techniques have been developed, employing immunofluorescent/epifluorescent microscopy, coupled with 5S rRNA sequencing, which allow accurate non-genetic detection of GEM. These techniques have been employed in aquatic systems. Charateristics of GEM important in release to the environment include ability to colonize surfaces, transfer genetic material and persist in specific environments. Clearly, the effects of GEM on the environment cannot be precisely predicted, unless the organisms have been so debilitated that they cannot persist in any natural habitat and cannot exchange genetic material with any other organism. It must be recognized that micro-organisms are extremely diverse and versatile. Uniformly applied, standard regulations governing deliberate release of GEM to the environment cannot be applied in the same way as for regulation of chemicals or medical devices. Case-by-case regulation appears to be the best approach for the immediate future. The implications of each organism, in terms of its own biology, will have to be considered.

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Resumen En el campo de la biotecnología, en rápida expansión, causa preocupación la liberación deliberada al medio ambiente de microorganismos manipulados genéticamente. Se estan llevando a cabo estudios para determinar el destino y el efecto de tales microorganismos en el medio ambiente y especialmente su impacto ambiental. Estos estudios indican que la detección directa de dichos microorganismos, no cultivables pero que permanecen viables en un entorno natural, se puede realizar empleando microscopía de inmunofluorescencia/epifluorescencia junto con secuencias 5S rARN utilizadas como etiquetas, lo que proporciona un sistema eficaz para rastrear estos organismos en muestras del medio ambiente. La utilización de microcosmos y los estudios realizados hasta el momento indican que, efectivamente, los microorganismos sufren una transformación que los mantiene viables aunque no recuperables, en un estado similar a la dormancia, por lo que se requieren métodos de detección directa para controlar la liberación al medio ambiente de microorganismos producidos mediante ingeniería genética. Este fenómeno ocurre también frecuentemente en otros microorganismos liberados al ambiente, entre ellos posibles patógenos. Los estudios realizados sobreVibrio cholerae proporcionan un ejemplo interesante tanto de los métodos empleados como de los resultados obtenidos. Las conclusiones obtenidas indican que el organismo esta ampliamente distribuido en el entorno natural, pero que solo puede ser detectado mediante microscopía directa debido al estado viable, aunque no vultivable, en que se mantiene en el ambiente. Ensayos en animales muentran que se pueden recolectar células deV. cholerae a partir de muestras del medio ambiente y recuperarse como células viables despues de haber sido inyectadas en el íleo de conejos, habiendose demostrado una respuesta ileal positiva para aquellas cepas que son enterotóxicas. Obviamente las ideas actuales acerca de la persistencia y función de los microorganismos en el ambiente necesitaran ser modificadas a medida que se vayan desarrollando nuevos métodos y nuevos conceptos en ecología microbiana.

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Résumé Le développement rapide de la biotechnologie a fait naître des préoccupations concernant le relâchement délibéré dans l'environment de micro-organismes génétiquement manipulés. Des études sont en cours pour déterminer le sort et les effects de ces micro-organismes dans la nature. Les travaux entrepris montrent que la microscopie d'immuno-fluorescence et d'épifluorescence, associée à la détérmination de séquences-marqueurs de r-ARN 5S, permet de détecter des micro-organismes non-cultivables, mais demeurés viables dans les milieux naturels. Les études déjà réalisées sur divers microcosmes indiquent que des micro-organismes peuvent effectivement se trouver dans un état viable, mais non-cultivable, qui ressemble à une dormance, ce qui exige, pour contrôler le relâchement dans la nature d'organismes génétiquement manipulés, l'emploi de méthodes de détection directe. Ce phénomène se produit fréquemment aussi dans le cas de beaucoup d'autres organismes, y compris des pathogènes potentiels, qui sont relâchés dans l'environnement. Des études réalisées avecVibrio cholereae fournissent un exemple de méthodes utiles et de résultats intéressants. Il résulte de ces études queV. cholereae est largement distribué dans l'environnement naturel et ne peut souvent y être détecté que par microscopie directe, à cause de l'état viable, mais non-cultivable, sous lequel il se maintient dans la nature. Les études sur l'animal montrent qu'on peut collecter à partir d'échantillons de milieu naturel des cellules deV. cholereae viables mais noncultivables, après avoir donné, dans le cas des souches entéro-toxigènes, une réponse intestinale positive. Il est clair que les idées courantes sur la persistance et la fonction des micro-organismes dans l'environnement demandent à être modifiées en fonction de l'émergence de nouvelles méthodes et de nouveaux concepts en écologie microbienne.

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Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki: 12–16.8.1985. Session 4     

The release of genetically engineered micro-organisms and viruses into the environment

This review considers the reasons for, and research governing, the regulation and monitoring of genetically engineered micro-organisms and viruses (GEMs) released into the environment. The hazards associated with releasing GEMs into the environment are the creation and evolution of new pests and diseases, and damage to the ecosystem and non target species. The similarities and differences between GEMs and conventional micro-organisms are discussed in relation to risk assessment. Other issues covered include the persistence of micro-organisms in the environment, transgene dispersal to non-engineered microbes and other organisms, the effects of transgenes and transformation on fitness, and the evolution of pests and pathogens that are given or acquire transgenes. Areas requiring further research are identified and recommendations for risk assessment made.     

The release into the environment of genetically engineered viruses, vaccines and viral pesticides

Viruses are being developed by genetic engineering procedures for two purposes: as improved vaccines or vectors for inserting foreign genes into a vaccinated hosts, and as improved viral pesticides. Both uses raise environmental issues.     

Assessing the welfare of genetically altered mice

In 2003, under the auspices of the main UK funders of biological and biomedical research, a working group was established with a remit to review potential welfare issues for genetically altered (GA) mice, to summarize current practice, and to recommend contemporary best practice for welfare assessments. The working group has produced a report which makes practical recommendations for GA mouse welfare assessment and dissemination of welfare information between establishments using a 'mouse passport'. The report can be found at www.nc3rs.org.uk/GAmice and www.lal.org.uk/gaa and includes templates for the recommended welfare assessment scheme and the mouse passport. An overview is provided below.     

Stress-induced evolution and the biosafety of genetically modified microorganisms released into the environment

This article is focused on the problems of reduction of the risk associated with the deliberate release of genetically modified microorganisms (GMMs) into the environment. Special attention is given to overview the most probable physiological and genetic processes which could be induced in the released GMMs by adverse environmental conditions, namely: (i) activation of quorum sensing and the functions associated with it, (ii) entering into a state of general resistance, (iii) activation of adaptive mutagenesis, adaptive amplifications and transpositions and (iv) stimulation of inter-species gene transfer. To reduce the risks associated with GMMs, the inactivation of their key genes responsible for stress-stimulated increase of viability and evolvability is proposed.     

Use of bioluminescence for detection of genetically engineered microorganisms released into the environment.

The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.     

Phenotypes of aquaporin mutants in genetically altered mice

The ability to move water across lipid membranes is crucial for nutrient intake, energy generation, waste excretion, and a myriad of other functions associated with life. Aquaporins, a family of integral membrane proteins, are now recognized as the channels responsible for transporting hydrophilic molecules, including water, across relatively impervious, hydrophobic cell membranes. A tremendous amount of work has been published on characterizing these proteins, which have been found in all bacteria, yeast, plants, and animals examined to date. In addition, an increasing number of mouse models with genetically altered aquaporin expression are being reported. This article will briefly review the basic biochemistry of aquaporins and then evaluate the use (and misuse) of mice in the quest for understanding the comparative pathophysiology of aquaporins in humans.     

Juvenile hypertension, the role of genetically altered steroid metabolism.

The importance of hypertension in the pediatric population is not as well appreciated as in adults. This might be related in part to the lower prevalence of high blood pressure in this age group. As with height and weight, blood pressure increases with age during childhood. The underlying causes of significant hypertension in children differ considerably from those in adults: while the prevalence of hypertension in pediatrics is lower than in adults, clinically identifiable causes of hypertension are common. Abnormalities in steroid biosynthesis have been known for years to cause hypertension in some cases of congenital adrenal hyperplasia. In these patients, hypertension usually accompanies a characteristic phenotype with abnormal sexual differentiation. Recently, the molecular basis of four forms of severe hypertension transmitted on an autosomal basis has been elucidated: (a) the glucocorticoid-remediable aldosteronism (GRA), (b) the syndrome of apparent mineralocorticoid excess (AME), (c) activating mutation of the mineralocorticoid receptor and (d) Liddle's syndrome. All these conditions are characterized primarily by low or low-normal plasma renin, normal or low serum potassium and salt-sensitive hypertension, indicating an increased mineralocorticoid effect. These forms of juvenile hypertension are a consequence of abnormal biosynthesis, metabolism or action of steroid hormones: (a) GRA is due to expression of a chimeric gene produced by fusion of 11beta-hydroxylase aldosterone-synthase genes. Expression of the chimeric enzyme occurs in the zona fasciculata of the adrenal cortex under the control of ACTH and can be suppressed by administration of glucocorticoids. (b) AME is caused by mutations of the 11beta-hydroxysteroid dehydrogenase type 2 enzyme, an enzyme that metabolizes cortisol into its receptor inactive keto-form cortisone, thus protecting the mineralocorticoid receptor (MR) from occupation by glucocorticoids. (c) The activating mutation of the MR results in constitutive MR activity and alters receptor specificity, with progesterone and other steroids lacking 21-hydroxyl groups becoming potent agonists. (d) Liddle's syndrome is due to mutations in the beta or gamma chain of the epithelial sodium channel in distal renal tubule cells. The hyperactivity of this channel caused by the mutations results in increased sodium reabsorption. With the advent of molecular biology in clinical practice it has become evident that some genetic defect may present with a more discrete phenotype, with only moderate hypertension with or without hypokalemia as presenting feature. Considering that hypertension in children and adolescents is often 'nonessential', a search for disorders should be integral part of the diagnostic work-up in young patients with hypertension.     

Biochemical characterization of a genetically altered calmodulin in Paramecium

Recent evidence proposes that the calcium-binding protein, calmodulin, plays a crucial role in the regulation or modulation of the calcium-dependent potassium conductance in Paramecium tetraurelia (Hinrichsen, R.D., Burgess-Cassler, A., Soltvedt, B.C., Hennessey, T. and Kung, C. (1986) Science 323, 503-506). We purified the calmodulins from both the wild type and pantophobiac A (a mutant lacking the above-mentioned conductance and whose phenotypic defect is traceable to its calmodulin) by hydrophobic interaction and immunoaffinity chromatographies, and examined them biochemically. In this paper we address the preliminary characterization of the two calmodulins and discuss the consequences of the genetic alteration. The differences described here are in their electrophoretic mobilities in polyacrylamide gel electrophoresis and in their binding characteristics to monoclonal antibodies raised against calmodulin from wild-type paramecia. Also, we present data which indicate a difference in the stimulation of the calmodulin-dependent enzyme bovine brain phosphodiesterase under certain conditions.     

Estrogen receptors in the kidney: Lessons from genetically altered mice

Background: Sex differences in human and animal models of kidney disease suggest that estrogen receptor (ER)-mediated events may modulate these processes. Genetically altered mice lacking one or both ERs provide a powerful tool to study these phenomena.Objective: This article examines sex differences in the kidney, particularly the role of ERs.Methods: To identify pertinent studies in genetically altered mice, a literature search was conducted on the MEDLINE database from January 1966 to July 2007, using the search terms estrogen receptor, kidney, and mice. Our group examined the effect of the ER-α knockout genotype on the kidney in streptozotocin-induced diabetes mellitus and compensatory kidney growth after uninephrectomy.Results: Female mice lacking ERa had reduced renal growth, including glomerular enlargement after 2 weeks of streptozotocin-induced diabetes mellitus and compensatory kidney growth 48 hours after uninephrectomy.Conclusion: ER-mediated events influence kidney growth and disease in female mice.     

The relevance of genetically altered mouse models of human disease

The impetus to develop useful models of human disease and toxicity has resulted in a number of large-scale mouse mutagenesis programmes. This, in turn, has stimulated considerable concern regarding the scientific validity and welfare of genetically altered mice, and the large numbers of mice that are required by such programmes. In this paper, the scientific advantages and limitations of genetically altered mice as models of several human diseases are discussed. We conclude that, while the use of some such mouse models has contributed considerably to an understanding of human disease and toxicity, other genetically altered mouse models have limited scientific relevance, and fewer have positively contributed to the development of novel human medicines. Suggestions for improving this unsatisfactory situation are made.     

Detected microsatellite polymorphisms in genetically altered inbred mouse strains

Microsatellites are 50–200 repetitive DNA sequences composed of 1- to 6-base-pair-long reiterative motifs within the genome. They are vulnerable to DNA modifications, such as recombination and/or integration, and are recognized as “sentinel” DNA. Our previous report indicated that the genotypes of the microsatellite loci could change from mono- to poly-morphisms (CMP) in gene knockout (KO) mice, implying that genetic modification induces microsatellite mutation. However, it is still unclear whether the random insertion of DNA fragments into mice genomes produced via transgene (Tg) or N-ethyl-N-nitrosourea (ENU) would also result in microsatellite mutations or microsatellite loci genotypes changes. This study was designed to find possible clues to answer this question. In brief, 198 microsatellite loci that were distributed among almost all of the chromosomes (except for the Y) were examined through polymerase chain reaction to screen possible CMPs in six Tg strains. First, for each strain, the microsatellite sequences of all loci were compared between Tg and the corresponding background strain to exclude genetic interference. Simultaneously, to exclude spontaneous mutation-related CMPs that might exist in the examined six strains, mice from five spontaneously mutated inbred strains were used as the negative controls. Additionally, the sequences of all loci in these spontaneous mutated mice were compared to corresponding genetic background controls. The results showed that 40 of the 198 (20.2 %) loci were identified as having CMPs in the examined Tg mice strains. The CMP genotypes were either homozygous or heterozygous compared to the background controls. Next, we applied the 40 CMP positive loci in ENU-mutated mice and their corresponding background controls. After that, a general comparison of CMPs that exist among Tg, ENU-treated and KO mouse strains was performed. The results indicated that four (D11mit258, D13mit3, D14mit102 and DXmit172) of the 40 (10 %) CMP loci were shared by Tg and KO mice, two (D15mit5 and D14mit102) (5 %) by Tg and ENU-treated mice, and one (D14mit102) (2.5 %) by all three genetic modifications. Collectively, our study implies that genetic modifications by KO, Tg or chemical mutant can trigger microsatellite CMPs in inbred mouse strains. These shared microsatellite loci could be regarded as “hot spots” of microsatellite mutation for genetic monitoring in genetic modified mice.     

The release of genetically modified crops into the environment. Part II. Overview of ecological risk assessment

Despite numerous future promises, there is a multitude of concerns about the impact of GM crops on the environment. Key issues in the environmental assessment of GM crops are putative invasiveness, vertical or horizontal gene flow, other ecological impacts, effects on biodiversity and the impact of presence of GM material in other products. These are all highly interdisciplinary and complex issues. A crucial component for a proper assessment is defining the appropriate baseline for comparison and decision. For GM crops, the best and most appropriately defined reference point is the impact of plants developed by traditional breeding. The latter is an integral and accepted part of agriculture. In many instances, the putative impacts identified for GM crops are very similar to the impacts of new cultivars derived from traditional breeding. When assessing GM crops relative to existing cultivars, the increased knowledge base underpinning the development of GM crops will provide greater confidence in the assurances plant science can give on the risks of releasing such crops.     

Safety assessment of genetically modified plants with deliberately altered composition

The development and marketing of ‘novel’ genetically modified (GM) crops in which composition has been deliberately altered poses a challenge to the European Union (EU)'s risk assessment processes, which are based on the concept of substantial equivalence with a non‐GM comparator. This article gives some examples of these novel GM crops and summarizes the conclusions of a report that was commissioned by the European Food Safety Authority on how the EU's risk assessment processes could be adapted to enable their safety to be assessed.