Environmental release of living modified organisms: current approaches and case studies

Agricultural biotechnology is being rapidly adopted as evidenced by the acreage of genetically modified (GM) crops planted and tonnes of product (grain and fiber) harvested. Concurrent with this technological progress, is a growing concern that the worlds biological diversity is coming under increasing threat from human activities. As such, ecological risk assessment approaches are being developed for GM crop plants as international agreements regulating the transboundary movements of these products are being implemented. This paper reviews the ecological risk assessment approach that has been used to date to approve GM crops to date. The process has been case-by-case, using a comparative, science-based approach balancing the potential risks and benefits of the new technology versus those present with the currently accepted practices. The approach used to evaluate and approve these products is consistent with the conditions and requirements outlined in the Cartagena Protocol.     

Environmental risks from the release of genetically modified organisms (GMOs)–the need for molecular ecology

Applications to release genetically modified organisms (GMOs) into the environment, usually the agricultural environment, are increasing exponentially. Many involve crop plants that are also weeds. Studies of biological invasions and of biological control show that the probability that a genetically new organism establishing itself is small; it is also unpredictable and in some cases could have severe ecological effects. GMOs pose risks both because they will be released in large numbers and because the greater the genetic novelty the greater the possibility of ecological novelty. Molecular ecology is an essential ingredient in ensuring that risks are assessed efficiently.     

Environmental mycobacteria in Korea. I. Distribution of the organisms

Environmental mycobacteria in Korea have been investigated by examining 54 soil, 111 house dust, 63 well water, and 98 sewage samples collected from 123 randomly selected areas in Korea during the fourth nationwide tuberculosis prevalence survey in 1980. A variety of mycobacteria were isolated from 76% of soil, 67% of sewage, 43% of well water, and 7% of house dust samples. Some samples yielded more than one species; thus 56 strains were obtained from soil, 107 strains from sewage, 48 strains from well water and 8 strains from house dust. Mycobacterium fortuitum was the most common species of environmental mycobacteria in Korea and the species was distributed equally in all types of samples tested. The M. terrae complex was also one of the common species of environmental mycobacteria and it seemed to be more abundant in water samples than in soil. Scotochromogenic slow growers M. scrofulaceum and M. gordonae were common microbes in soil and water samples, although the latter was more frequently detected in water samples. Scotochromogenic rapid growers M. flavescens and M.phlei, and photochromogenic rapid grower M. vaccae were isolated more frequently from sewage or water samples than from soil. Nonphotochromogenic rapid growers M. chelonei (chelonae) and M. smegmatis were isolated mostly from sewage and the former was rarely found in soil and well water samples. The clinically important species M. avium-intracellulare complex was found less frequently in all types of test samples.     

Key issues in the deliberate release of genetically-manipulated bacteria

Abstract The deliberate release of a genetically engineered bacterium often requires that a complex pathway be travelled through scientific and regulatory questions. It is important to consider the scientific aim of the release and the nature of the modification (deletion or insertion, site of insertion, level of expression) and its likely effect on survival of the organism and the possibility of gene transfer. In Australia, the Genetic Manipulation Advisory Committee assesses applications and makes recommendations about pre-release testing and procedures for conducting field release. Two examples of field release of genetically manipulated bacteria in Australia are considered. Firstly, the commercial product Agrobacterium strain K1026 (‘NoGall’^TM), a genetically engineered biological control agent for crown gall disease of stone fruits and roses. Secondly, a lacZY -marked derivative of a strain of Pseudomonas corrugata , that can act as a biological control agent against take-all disease of wheat. Prior to release, bacterial survival and competition was tested in soil microcosms. The distribution and survival of the organism were monitored after field release. Since 1992 the marked bacteria have been recovered only after enrichment. Assessment of risk should consider the survival and spread of the genetically manipulated bacterium and its foreign DNA and the impact of the inoculated bacteria on other (‘non-target’) organisms.     

Safety standards for the environmental release of genetically engineered organisms

The use of genetically engineered organisms holds considerable promise for environmental management and other purposes, provided appropriate safety standards are established and observed. The problem with much of the debate concerning deliberate releases has been the difficulty in getting down to specifics. Examples can be advanced that give cause for concern, or that demonstrate that introductions can be carried out safely; but none of these has the generality to apply to all cases. Generic arguments for and agaisnt the safety of introductions must be rejected, and replaced by consideration of the properties of individual introductions. It is the properties of the introduced organism in relation to the environment that must receive attention, not the method by which the genetic modification was achieved. One must go beyond discussions that lump all possible applications together, and develop criteria that associate individual cases with the risk categories most appropriate to them.     

转基因微生物生态学及大田释放风险评价研究

向环境中释放转基因微生物可能会带来一系列的安全问题．在大面积释放之前必须对转基因微生物在环境中发生基因转移的潜力、存活能力、扩散能力及对生态系统的潜在影响等进行生态学研究和风险评价,同时还要探索有效的检测方法和风险评价策略．该研究有助于分子生态学的发展和生物技术的安全应用,具有重要的理论和实践意义．     

Combinational biosynthesis of phycocyanobilin using genetically-engineered Escherichia coli

Genes of the key enzymes for phycocyanobilin (PCB) biosynthesis were cloned into E. coli and combinationally expressed to produce phycocyanobilin, with autologous heme as substrate. Culture conditions were optimized to achieve ~3 mg PCB/l. A protocol for the purification of recombinant phycocyanobilin was established using solvent extraction combined with chromatography, which resulted in a final yield of ~0.3 mg PCB/l with a purity >95 %. Recombinant phycocyanobilin could scavenge hydroxyl radicals with an EC₅₀ of 0.1 μM.     

Effect of temperature and plasmid carriage on nonculturability in organisms targeted for release

Abstract: The ability to track genetically modified bacteria released into the environment is essential for assessing their persistence and dispersal. Some bacteria can enter a 'viable but nonculturable' (VBNC) state in which the cells remain viable while losing the ability to grow on routine culture media. Thus, VBNC cells are not detectable by standard plating methods. In order to determine what conditions, if any, induce this state in Pseudomonas fluorescens, Pseudomonas syringae , and Escherichia coli , cells were 'marked' with lux genes, either chromosomally or on one of two different plasmids. Variations in temperature, but not nutrient or NaCl concentrations, affected culturability of these strains and induced the VBNC state. The temperature which induced the VBNC state in the two pseudomonads depended on whether or not the cell carried one of the two lux -marked plasmids. This effect was shown not to be due to the presence of the lux genes, as their removal from the plasmid had no effect on entry into the VBNC state. Instead, the effect appeared to depend on the location of the plasmid DNA, as a strain of P. fluorescens with the same plasmid integrated into the chromosome behaved identically to the parent strain. The fact that plasmids may have such a dramatic effect on culturability has significant implications for the monitoring of genetically modified bacteria intended for environmental release.     

Establishing relative release kinetics of faecal indicator organisms from different faecal matrices

Aims: A laboratory assay for comparative characterization of various faecal matrices with respect to faecal indicator organism (FIO) release using, artificial rain water. Methods and Results: Fresh sheep and beef‐cattle faeces, dairy cattle slurry and beef cattle farm yard manure (FYM) were collected from commercial units in south‐west England and applied to 20 randomized 1 m² plots established on permanent grassland. Representative samples from each faecal matrix (n = 5) were collected on four occasions over 16 days. One gram of each sample was transferred to a sterile vial to which 9 ml of standard local rain was carefully pipetted. The vial was then rotated through 360°, 20 times in 60 s to ‘simulate’ a standardized interaction of the faecal material with rainfall, providing an assay of comparative release potential. Appropriate decimal dilutions were prepared from the eluent. Following agitation, with a sterile spatula, the remaining faecal material and eluent in the vials were vortex mixed for 60 s before decimal dilutions were prepared from the resulting mixture, providing a quantitative assessment of the total FIO in the sample from which percentage release could be determined. Bacterial concentrations were enumerated in duplicate by membrane filtration following standard methods for FIO. Significant differences in release kinetics of Escherichia coli and enterococci from each of the faecal matrices were determined. Conclusions: Differences in release from each faecal substrate and between FIO type (E. coli and intestinal enterococci) were observed in this laboratory study. The order of release of E. coli from the faecal matrices (greatest to least, expressed as a percentage of the total present) was dairy cattle slurry > beef cattle FYM > beef‐cattle faeces > sheep faeces. For intestinal enterococci the order of percentage release was dairy cattle slurry > beef‐cattle faeces > beef cattle FYM > sheep faeces. Significance and Impact of the Study: This laboratory‐based method provides the first data on the relative release kinetics of FIO from different faecal matrices in rain water. This is fundamental information needed to parameterize laboratory‐based microbial models and inform approaches to field and catchment risk assessment.     

Environmental constraints upon locomotion and predator-prey interactions in aquatic organisms: an introduction

Environmental constraints in aquatic habitats have become topics of concern to both the scientific community and the public at large. In particular, coastal and freshwater habitats are subject to dramatic variability in various environmental factors, as a result of both natural and anthropogenic processes. The protection and sustainable management of all aquatic habitats requires greater understanding of how environmental constraints influence aquatic organisms. Locomotion and predator-prey interactions are intimately linked and fundamental to the survival of mobile aquatic organisms. This paper summarizes the main points from the review and research articles which comprise the theme issue 'Environmental constraints upon locomotion and predator-prey interactions in aquatic organisms'. The articles explore how natural and anthropogenic factors can constrain these two fundamental activities in a diverse range of organisms from phytoplankton to marine mammals. Some major environmental constraints derive from the intrinsic properties of the fluid and are mechanical in nature, such as viscosity and flow regime. Other constraints derive from direct effects of factors, such as temperature, oxygen content of the water or turbidity, upon the mechanisms underlying the performance of locomotion and predator-prey interactions. The effect of these factors on performance at the tissue and organ level is reflected in constraints upon performance of the whole organism. All these constraints can influence behaviour. Ultimately, they can have an impact on ecological performance. One issue that requires particular attention is how factors such as temperature and oxygen can exert different constraints on the physiology and behaviour of different taxa and the ecological implications of this. Given the multiplicity of constraints, the complexity of their interactions, and the variety of biological levels at which they can act, there is a clear need for integration between the fields of physiology, biomechanics, behaviour, ecology, biological modelling and evolution in both laboratory and field studies. For studies on animals in their natural environment, further technological advances are required to allow investigation of how the prevailing physico-chemical conditions influence basic physiological processes and behaviour.     

Distribution of a genetically-engineered Escherichia coli population introduced into soil

The spatial localization of the cells and the DNA of a genetically-engineered Escherichia coli population introduced into soil was investigated. Inoculated soils were size fractioned and bacterial numbers and E. coli EL 1003 specific chromosomal DNA target sequences were enumerated in each fraction using plate-counting and MPN-PCR, respectively. Different numbers of either indigenous or introduced bacteria were found in each fraction indicating that their distribution in the soil was non-uniform. The distributions of the indigenous bacteria and the E. coli cells within the size fractions were significantly different: the E. coli population was mainly associated with the dispersible clay fraction (79·0%) from which only 10·7% of the indigenous bacteria were recovered. The distribution of the E. coli target DNA sequences was in agreement with the location of the cells. The different distribution of the two populations is likely to restrict genetic interactions. These results are relevant to potential interactions between native soil microflora and populations introduced into soil for competitive purposes.     

神府东胜煤田开发中诱发的环境灾害问题研究

神府东胜煤田地处晋陕蒙水蚀风蚀交错的生态脆弱带 ,开发中出现的环境问题已威胁到矿区生产安全。大量弃土弃渣堆积于河道与沟道 ,加剧了水土流失 ;大柳塔等矿发生 8hm2 的塌陷与地裂缝 ,引起地下水渗漏 ,水位下降 2～ 3m ,甚至断流 ;大量植物死亡 ,土地沙化日趋严重 ;人为滑坡、泥石流活动频繁 ;河道泥沙变粗近 5倍 ,粗沙比例增加 ,淤积使过水断面减少了 37.6 % ,行洪能力降低了 2个数量级。通过对弃土弃渣集中排放 ,扰动土回填复垦 ,植树造林的措施建立矿区环境整治技术体系 ,根本改变生态环境使矿区生产持续稳定地发展     

Application of genetically-engineered anti-CEA antibodies for potential immunotherapy of colorectal cancer.

Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors, and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for "humanization" of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or used for therapeutic strategies which similarly take advantage of the ability of the antibodies to target cytotoxic agents selectively to tumor cells. The application of these novel procedures for manipulating MAb structure presents entirely new opportunities for diagnosis and treatment of human colorectal cancer.     

Mutation and screening to increase chymosin yield in a genetically-engineered strain ofAspergillus awamori

Summary Through the course of five rounds of mutagenesis of a genetically-engineered strain ofAspergillus awamori, the yield of a heterologous protein (the acid protease, calf chymosin) increased four-fold. This was accomplished through the use of an agar plate screen incorporating the colony restrictor 2,6-dichloro-4-nitroaniline (dichloran) and the acid protease inhibitor diazoacetyl-norleucine methyl ester (DAN) to reduce high background concentrations of the native acid protease. A miniaturized liquid culture growth method using 24-well culture plates was an intermediate screen between agar plate and shake flask cultures. Analysis of broth samples for active calf chymosin was accomplished with a highly specific, 96-well microtiter plate turbidimetric assay.