INFORMATION SYSTEMS FOR BIOTECHNOLOGY


February 2002

COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:
Potential Environmental Risks and Hazards of Biotechnology
Transgenes—By No Easy Means
Phytoremediation Of TNT
FAO Listserv



POTENTIAL ENVIRONMENTAL RISKS AND HAZARDS OF BIOTECHNOLOGY
Part II: Methods to Estimate Risks and Hazards

Introduction
In Part I, William Muir and his associate Richard Howard conclude that the risk of releasing transgenic organisms to the environment can be assessed by addressing the probability of exposure to the hazard, P(E), rather than the probability of harm given exposure, P(H/E) (see ISB News Report, November 2001,
http://www.isb.vt.edu/news/2001/news01.nov.html#nov0105 ). The probability of exposure is equal to the expected long-term outcome of natural selection for the transgene, given that the transgene has escaped into a natural environment. Although escape results in initial exposure, harm ultimately results from long-term exposure—the transgene may increase in frequency until it becomes the norm rather than the exception (P(E)=1), or, with time, the transgene will be culled from the population (P(E)=0). The exception occurs if there is a massive escape and/or the wild population is very small.

Natural selection occurs as a result of the differential ability of genotypes to produce offspring for the next generation. This differential ability is termed overall or net fitness. Muir and Howard have reduced overall fitness to six components: juvenile and adult viability, age at sexual maturity, female fecundity, male fertility, and mating success.1,2,3 They then incorporated these components into a mathematical model that integrates them into a single prediction of risk. The discussion that follows will explain how the fitness components are estimated and demonstrate use of the model using Japanese medaka fish.

Estimation of Fitness Components
Terminology

The model is based on the assumption that transgene expression is completely dominant and that individuals hemizygous for the transgene (Tw) have the same phenotype as homozygous (TT) individuals. Heterozygous individuals are termed hemizygous because there is no complementary allele for the transgene. Nevertheless, the absent allele at that locus may be represented as w and the transgene allele may be represented as T. In the following notation, the transgenics' genotypes TT and Tw are designated as subscripts 2 and 3, respectively.

Juvenile Viability (vj )
Juvenile viability is simply defined as survival from the embryo to the age of sexual maturity (or approaching sexual maturity). There are several ways to estimate this component. The simplest experiment would be to establish two pure breeding lines (TT and ww) and, starting with a known number of fertile eggs, count the number that survive to sexual maturity. The experiment should be conducted under environmental conditions that closely approximate the natural environment into which they might escape. This experiment should be replicated several times (2 to 10). The average percent survival of each genotype (v'j) is converted to per day viabilities (vj) occurring between consecutive census time periods (at+1 and at) by assuming a log-linear reduction in daily viability between time periods. Viability can then be described by the following equation.

For example, if 10 replicates of 1000 fry results in an average of 175 transgenic and 250 wild-type individuals surviving to 56 days of age, the per day survival rate is calculated thus:

An alternative method that addresses the issue of background genotype, but does not require the offspring to be genotyped, is given by Muir and Howard.1,2 This method is based on the theory that the only difference between survival of an intercross and a backcross is the segregation ratio of 3:1 vs. 1:1. If the viabilities of each genotype are the same, then the expected survival for each cross would be the same. If viability of the transgenic genotype is less than that of wild type, then total survival of the intercross will be less than that of a backcross. Preferably the wild-type line is representative of the native fish in the area into which the fish might escape. In this way, the background genotype of both transgenic and wild-type fish are taken into account.

The procedure is to cross the homozygous transgenic line with a wild-type strain to produce the F1 generation. The cross F1 is then intercrossed to produce the F2 generation and the F1 is also backcrossed to the wild type stock to produce the BC1 generation. The number of fish that survive from hatching to sexual maturity (or approaching sexual maturity) is recorded. The relative viabilities are then found by the method of maximum likelihood as shown by Muir and Howard.1

Adult Viability (uj )
Ideally, adult survival of each genotype would be measured in as natural an environment as possible until most fish die. Because this may take a very long time, an alternative method is to assume a log-linear reduction in daily viability and observe only enough time periods to establish a trend.

For example, assume 1000 fish of each genotype are observed for 100 days past sexual maturity. The proportion surviving at the termination of the experiment of each genotype (u'j) is 90% for wild-type and transgenic individuals. The daily reduction in survival (uj) is determined using the following equation:

In our example, at+1- at = 100 days. Thus

Age at Sexual Maturity (sj )
Age at sexual maturity can be straightforward or difficult, depending on the species. For medaka, age at sexual maturity was recorded as the age at which females first produced eggs. We assumed the males to be mature at the same age. For other species, it may be necessary to sacrifice the animals at various ages and observe gonadal development. Assume for this example that ages at sexual maturity are 56 and 49 days after hatching for wild type and transgenic fish, respectively.

Female Fecundity (cj )
Fecundity is also straightforward. For medaka, estimating fecundity is a simple matter of counting the number of eggs produced from each genotype. The genotypes should be the same age and several fish should be measured. For this example, assume wild-type fish produce an average of 8.8 eggs per spawn and transgenic produce 11.4.

Male Fertility (rj )
Fertility is more difficult to determine than fecundity and is inferred from the number of eggs fertilized by alternative genotypes. We examined the ability of the male genotype to fertilize eggs with a simple, completely randomized design experiment in which 10 transgenic males and 10 wild-type females were randomly single-pair mated with wild-type females in separate 40-liter tanks for eight days. The first three egg masses produced by each female in that eight-day period were collected and incubated in a hatching tank. Twenty-four hours later, the eggs were examined under a dissecting microscope and classified as fertile or infertile based on presence or absence of embryo development. Assume for this example that both wild-type and transgenic males have fertility rates of 95%.

Relative Mating Success (mj ) and (fi )
As with juvenile viability, mating success of each genotype can be determined either directly or indirectly. Direct observation is the simplest, but may not be possible in all species. With direct observation, staged mating trials are conducted that allow for both mate competition and mate choice. The mating frequency of each genotype is recorded.4,5 Because the effect of the transgene on mating success can vary with its relative frequency, such trials should also be performed using different ratios of genotypes.

With indirect observation, transgenic and wild type males and females in various ratios are placed together in a setting and allowed to mate. Muir and Howard2 call these trials `mating sets.' Several different mating sets, and replications of mating sets, are conducted. After mating, the identity of the male parent is inferred from the genotypes of the progeny. In the simplest case, a single wild-type female would be introduced into a tank containing a transgenic and wild-type male. Any transgenic offspring would immediately identify the genotype of the male parent. Assume for this example an equal mating success of transgenic and wild-type males and females.

The Model and Prediction of Exposure
Table 1 presents a summary of the estimates for net fitness components of Japanese medaka.

Table 1.
Table 1. Fitness component Wild type (ww) Transgenic (TT,Tw)
Juvenile survival to sexual maturity (v'j) 25.0% 17.5%
Daily juvenile viability (v'j) 0.9755 0.9649
Adult survival 100 days past sexual maturity (u'j) 90% 90%
Adult viability (uj) 0.9989 0.9989
Male fertility (rj) 95% 95%
Relative male mating success (mj) 100% 100%
Relative female mating success (fi) 100% 100%
Female fecundity (eggs/clutch) (cj) 8.8 11.4
Age at sexual maturity (si) 56 days 49 days

The model, which incorporates all these effects, is given by Muir and Howard1,2 and is programmed to allow for any combination of fitness components. An interactive trial model is available at http://www.isb.vt.edu/nfca/nfca1.cfm.

Before starting to calculate fitness, one usually assumes a stable wild-type population age distribution. The initial age distribution is set using an exponential decay parameter `b'. The value of this parameter is found by trial and error—the fitness values are set for transgenic fish the same as for wild-type, i.e., the initial population consists only of wild-type fish, or at least fish that do not differ in their fitness. The value of b is found such that the initial and stable age distribution, determined after many generations, is similar. This parameter is not critical to the program, but establishes at least a reasonable starting distribution. Using these above components, the constant value of b for a stable age distribution was found to be 0.93.

Assuming an initial population size of 60,000 and that 60 transgenic individuals were introduced at 56 days of age, Figure 1 gives the predicted gene frequency over the first 40 generations. The increase in transgene frequency suggests a high risk for spread of the transgenic organism, i.e., the transgene will eventually become fixed.

Sources

1. Muir WM and Howard RD. 2001. Fitness components and ecological risk of transgenic release: A model using Japanese medaka (Oryzias latipes). American Naturalist 158: 1-16.

2. Muir WM and Howard RD. 2002a. Methods to assess ecological risks of transgenic fish releases. In Genetically Engineered Organisms: Assessing Environmental and Human Health Effects, eds. DK Letourneau and BE Burrows, 355-383. CRC Press.

3. Muir WM and Howard RD. 2002b. Environmental risk assessment of transgenic fish with implications for other diploid organisms. In press.

4. Hallerman E and Kapuscinski A. 1993. Potential impacts of transgenic and genetically manipulated fish on natural populations: Addressing the uncertainties through field testing. Genetic Conservation of Salmonid Fishes, eds. JG Cloud and GH Thorgaard. New York: Plenum Press.

5. Howard RD et al. 1998. Mate choice and mate competition influence male body size in Japanese medaka. Animal Behaviour 55: 1151-1163.

William M. Muir
Departments of Animal and Biological Sciences
Purdue University
bmuir@purdue.edu



TRANSGENES—BY NO EASY MEANS

The current widespread application of genetic engineering to crop species is largely due to the ease of plant transformation. Plant transformation, the process of introducing a foreign or engineered DNA element into the native genome of a plant, has been successfully performed for almost 20 years. However, due to a lack of understanding of the underlying molecular mechanisms of transgene introduction and integration, plant transformation remains more an art than a science. All of the three main techniques used for plant transformation, Agrobacterium-mediated, protoplast, and particle bombardment transformation, result in unpredictable integration of transgenes. This has led to concerns that transformation might indirectly alter the expression of other genes, resulting in a toxic or allergenic phenotype. Fortunately, recent research is expanding our understanding of how introduced genes are integrated at the molecular level during transformation, suggesting strategies for controlling the location and expression of transgenes.

In any plant transformation experiment, the researcher knows that many independent transgenic lines will have to be screened before a line stably expressing a single copy of the transgene is isolated. Frequently, many transgenic plants will contain multiple copies of the transgene, either in the form of tandem repeats at a single locus, or scattered throughout the genome of the plant. This is a problem for two reasons. First, the integration of multiple copies of a transgene has been linked with gene silencing, a poorly understood phenomenon, where the expression of an introduced gene is somehow detected and "shut off" by the plant's cellular machinery. Second, overexpression of the transgene due to multiple copies can prove to be toxic to the plant, leading to poor growth or even no growth. Even if the plant contains only a single copy of the gene, there is no guarantee that it will be expressed correctly. The degree of expression of the transgene can also be determined by the site of insertion, otherwise known as the "positional effect."

The main techniques used for plant transformation can be loosely grouped under two headings: Agrobacterium-mediated transformation and methods that use direct DNA delivery for transformation. Protoplast transformation was the first plant transformation technique developed using direct DNA delivery. In this method, protoplasts derived either directly from plant tissues or from a plant cell suspension culture are induced to take up naked DNA through treatment with membrane permiabilization agents such as polyethylene glycol (PEG) or by electroporation. The method is useful, as it is genotype-independent, but the degree of finesse required for success, along with the high occurrence of spontaneous mutations caused by long periods in tissue culture, restrict the application of the technique to species recalcitrant to other methods of transformation.

A more commonly used method of direct DNA delivery transformation is a method known as microprojectile, particle bombardment, or biolistic transformation. In this method, tiny particles of tungsten or gold are coated with DNA containing the construct of interest. These particles are then "shot" into the plant tissue using gunpowder, gas, compressed air, or other methods of acceleration. The force of the acceleration drives the tiny particles through the wall and membrane of the plant cells, delivering the naked DNA directly into the cells' interiors. The exact mechanism of how the naked DNA then becomes integrated into the plant's genome is unknown, but multiple studies have shown that in the vast majority of cases microprojectile transformation results in the integration of multiple, often rearranged, copies of the transgene. One currently proposed theory suggests that the introduced transgenes are first spliced into arrays by the cells' endogenous machinery before integration into the plant's genome. This theory might partially explain the main drawback of this transformation method—the high occurrence of genetic rearrangements found in recovered transformants.

Another explanation may be found in the results of a study, reported in Theoretical and Applied Genetics, indicating that microprojectile transformation may involve chromosome breakage and re-ligation. In this study, Svitashev et al. characterized transgenic lines of hexaploid oat, using a combination of phenotype, genotype segregation, Southern blot, and fluorescence in situ hybridization (FISH) analyses.1 Six of the 25 transgene loci examined were associated with rearranged chromosomes. Through Southern blot analysis and FISH performed on metaphase chromosomes, evidence of both chromosomal rearrangement and breakage events could be detected. The authors theorize that this may be the result of physical breakage of the host cell's DNA during particle bombardment or, possibly, the integration event itself. However, these results conflict with the data described in a second, more recent paper in the same journal. Jackson et al. studied 13 independent transgenic wheat lines transformed using microprojectile bombardment.2 The authors used a high-resolution form of FISH to physically map the location and structure of the integrated transgenes. Although the authors found evidence of large, tandem repeats of the transgenes integrated in the plant's genome, they were unable to detect any chromosomal rearrangements associated with the integrated transgenes. Regardless of the exact nature of the mechanism, it seems clear from the data described in these papers that microprojectile transformation often results in transgenic plants with a complex pattern of transgene integration.

Agrobacterium-mediated transformation, the most widely used method of plant transformation, utilizes the natural ability of the plant pathogen, Agrobacterium tumefaciens, to transfer DNA sequences from a particular segment of an endogenous plasmid within the bacterium to the nuclear genome of the plant. This segment, known as the T-DNA, usually includes one or two genes of interest, as well as a marker gene. This method is widely favored due to its ease of use and low cost. Unfortunately, the restricted host range of the bacterium meant that some dicot and most monocot species were, until recently, incompatible with the technique. However, the development of new, supervirulent forms of the plasmid vector and species-specific pretreatments has led to a dramatic expansion in the number of species transformed using this technique. Another reason for the popularity of this method is that the T-DNA usually seems to integrate in transcriptionally active regions of the plant genome, increasing the likelihood that the transgene will be expressed.

However, it is not uncommon for Agrobacterium-mediated transformation to result in the integration of multiple copies of the transgene in the form of tandem repeats. Tandem repeats resulting from T-DNA insertion have been reported and investigated in a number of crop species. These types of repeats can be difficult to detect by Southern blot, since they tend to integrate at a single location in the plant genome. In a recent paper published in Molecular and General Genetics, Kumar and Fladung reported using rpPCR, a method that utilizes primer pairs oriented in opposite directions, to identify tandem repeats in 45 transgenic aspen and hybrid aspen lines transformed with six different constructs.3 All the transgenic lines were generated through standard Agrobacterium-mediated transformation. In the lines examined, 21% contained multiple repeats of the transgene; however the organization of the repeats consisted of both direct and inverted repeats. Some of the lines were also found to contain "filler" DNA between the repeated T-DNAs, ranging from four to almost 300 base pairs. Interestingly, the authors found that all of the direct repeats contained identical residual right-border repeat sequences. They speculate that this sequence, combined with the mechanism of T-DNA insertion, is responsible for the formation of direct repeats. However, a single mechanism is unlikely to account for all the different repeat structures seen resulting from Agrobacterium-mediated transformation.

Currently, transgene integration into the host genome is essentially random, regardless of the method used to perform the transformation. As a result, attempts have been made to develop a system for targeted transgene insertion, either through the use of scaffold attachment sites or through the introduction of elements of a homologous recombination system, such as the Cre/lox system. To date, however, these efforts have yielded inconsistent results, making them unsuitable for commercial application. Nevertheless, these technologies are making great advances, and it is hoped that, combined with the increasing understanding of the mechanisms of transgene integration, it will soon be possible to precisely and consistently engineer plants to express a single copy of an introduced gene.

Sources

1.  Svitashev S, Ananiev E, Pawlowski WP, and Somers DA. 2000. Association of transgene integration sites with chromosome rearrangements in hexaploid oat. Theoretical and Applied Genetics 100: 872-880.

2.  Jackson SA, Zhang P, Chen WP, Phillips RL, Friebe B, Muthukrishnan S, and Gill BS. 2001. High-resolution structural analysis of biolistic transgene integration into the genome of wheat. Theoretical and Applied Genetics 103: 56-62.

3. Kumar S and Fladung M. 2000. Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Molecular and General Genetics 264: 20-28.

Claire Granger
Biologist
alesia_sun@yahoo.com


PHYTOREMEDIATION OF TNT

Until now, bacteria have been the preferred organisms for in situ bioremediation of hazardous wastes. Bacteria, particularly many species of the soil borne Pseudomonas, were favored because they possess a wide array of enzymes capable of degrading complex organic compounds found in industrial wastes. Phytoremediation, which is bioremediation using plants, is proving to be another powerful strategy for safely removing simple organic compounds from contaminated soil and water (refer to http://aec.army. mil/prod/usaec/et/restor/phyto_01.htm). Wetlands planted with a variety of plants can clean the wastes from sewage effluent, making the water nearly drinkable without further treatment. The natural ability of some plants to take up heavy metals and radioactive elements is also being exploited for certain bioremediation efforts.

Some phytoremediation involves the cooperation of bacteria and fungi living in the rhizosphere. It is these organisms, not the plants themselves, that carry out the chemical degradation of wastes. However, this tactic poses many challenging problems. It is difficult to find the right combination of environmental and rhizosphere conditions conducive to bioremediation.1

Contamination by complex organic compounds is particularly difficult to treat with phytoremediation because of two major problems: 1) the high toxicity of the compounds and their degradation products, and 2) the paucity of plant enzymes capable of safely degrading the compounds. Despite these difficulties, investigators are pursuing the phytoremediation of aromatic compounds such as explosives.
 
Victor Medina, Washington State University - Tricities Branch, is studying phytoremediation of 2,4,6-trinitrotoluene (TNT) and related compounds through a series of projects investigating organic and inorganic compound remediation.2 Medina's work shows that certain plant roots have the enzymatic capability for reducing TNT into non-explosive transformation products that are assimilated into the plant material.

The US Army investigated using phytoremediation to remove explosives from contaminated wetlands using various aquatic and wetland plants ( http://www.wes.army.mil/el/resbrief/phytoexp.html). In 1999 they reported that although the uptake and biotransformation rates of radioisotope-labeled TNT and RDX from the wetland systems were high, complete biodegradation to CO2 was very low. The army research was hoping to achieve "ultimate" biodegradation, which is the decay of the natural and xenobiotic organic chemicals into small molecules such as carbon dioxide and water. In contrast, "primary" and "environmentally acceptable" degradation involves loss of the chemical's toxicological activity.

Neil C. Bruce and colleagues, at the University of Cambridge, have recently reported using transgenic tobacco plants expressing a bacterial enzyme to detoxify TNT. 3 They isolated the nitroreductase gene, nfsI, from Enterobacter cloacae, which has been found to be active against TNT, and modified it to contain a consensus start sequence to facilitate translation. The construct was then inserted into tobacco (Nicotiana tabacum cv. Xanthi) leaf discs using an Agrobacterium transformation system. The T1 generation of successful transformants was allowed to self-fertilize and the most TNT-tolerant transgenic line, designated NR3-2, was selected for the TNT toxicity and transformation studies.

Duplicate batches of seeds from both wild-type and transgenic lines were germinated and grown in liquid medium for 14 days and then transferred to media containing either 0, 0.1, 0.25, or 0.5 mM TNT. Transgenic plants showed a greater biomass weight gain and better survivability than wild-type plants in the TNT media at all levels. For example, at the 0.25 mM TNT level, wild-type plants lost 34% of their wet weight, while NR 3-2 plants gained 18%. Growth was similar for both groups in the absence of TNT. The removal of TNT and its metabolites in the growth medium was measured by HPLC. Both transgenic and wild-type plants removed TNT from the medium, however, at greatly differing rates. At the 0.1 mM TNT level, the wild-type plants removed 78% of the TNT in 168 hours, but the NR 3-2 transgenic line removed 71% in six hours, with total removal achieved within only 20 hours. The concentration of TNT in the seedlings was also examined. Both roots and shoots of the wild-type lines contained TNT and its metabolite ADNT (aminodinitrotoluene). ADNTs are an undesirable metabolite thought to be toxic and possibly carcinogenic. No TNT was extracted from tissues of the transgenic line and only trace amounts of ADNT were found in the roots, indicating either the complete degradation of absorbed TNT or the sequestering of TNT in a non-extractable form. As an explanation, the authors referred to reports that indicate xenobiotic compounds in plants may be conjugated to various compounds and then compartmentalized in the vacuole as lignin, rendering them harmless.

A variety of naturally occurring soil bacteria possess a wide array of enzymes capable of degrading insecticides and herbicides. Pseudomonas is the workhorse of the bioremediation bacteria. They can break down polycyclic aromatic hydrocarbons styrene and TNT. Some other superior bioremediators include Burkholderia cepacia, which degrades polychlorinated biphenyls, Enterobacter cloacae, which decomposes TMT and RDX, Dehalococcoides ethenogenes, a degrader of trichloroethene, and Sphingomonas aromaticivorans, which decomposes fluorine, naphthalene, toluene, and xylene. A recognized pesticide-degrading bacterium is Desulfitobacterium hafniense.

Many algae, bacteria, and fungi are already exploited for remediation of complex molecules chemically related to agricultural chemicals. Genomic information on these organisms is gathered by the US Department of Energy and used to search for bioremediation enzymes ( http://www.ornl.gov/microbialgenomes/organisms.html). However, microbes alone are not always adequate for reducing pesticide and herbicide residues to acceptable levels within a growing season and may produce toxic intermediates capable of being taken up by plants. A complex concoction of organisms is usually needed to ensure ultimate remediation. Bruce's transgenic tobacco plants offer advantages because they may exceed the utility of current bacterial applications in bioremediation and did not show evidence that they accumulate toxic residues in the plant tissues.

Sources

1. Anderson TA, Gutherie EA and Walton BT. 1993. Bioremediation in the rhizosphere. Environmental Science and Technology 27(13): 2360-2636.

2. Medina VF, Larson SL, Bergstedt AE, and McCutcheon SC. 2000. Phyto-removal of trinitrotoluene from water using batch kinetics studies. Water Research 34(10): 2713-2722.

3. Hannink N, Rosser SJ, French CE, Basran A, Murray JAH, Nicklin S and Bruce NC. 2001 Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase. Nature Biotechnology 19(12): 1168-1172.

Brian R. Shmaefsky
Department of Biology and Environmental Sciences
Kingwood College
brian.shmaefsky@nhmccd.edu



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