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PubMed2006

Genetically modified baculoviruses: a historical overview and future outlook.

The concept of using genetic engineering to improve the natural insecticidal activity of baculoviruses emerged during the 1980s. Both academic and industrial laboratories have since invested a great deal of effort to generate genetically modified (GM) or recombinant baculoviruses with dramatically improved speeds of kill. Optimal production methodologies and formulations have also been developed, and the safety and ecology of the recombinant baculoviruses have been thoroughly investigated. Unfortunately, the initial excitement that was generated by these technologies was tempered when industry made a critical decision to not complete the registration process of GM baculoviruses for pest insect control. In this chapter, we summarize the developments in the field from a historical perspective and provide our opinions as to the current status and future potential of the technology. We will argue that GM baculoviruses are valuable and viable tools for pest insect control both alone and in combination with wild-type viruses. We believe that these highly effective biopesticides still have a bright future in modern agriculture as public awareness and acceptance of GM organisms, including GM baculoviruses, increases.

Animals↗
PubMed2005

Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein.

The ability of dendritic cells (DCs), genetically modified with one of two types of plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses, is compared. The first type, also called "secreted" vaccine (sVac), encodes for the full length of the human prostate-specific antigen (PSA) with a signal peptide sequence so that the expressed product is glycosylated and directed to the secretory pathway. The second type, truncated vaccines (tVacs), encodes for either hPSA or human prostate acidic phosphatase (hPAP), both of which lack signal peptide sequences and are retained in the cytosol and degraded by the proteasomes following expression. Monocyte-derived dendritic cells are transiently transfected with either sVac or one of two tVacs. The DCs are then used to activate CD25+-depleted or nondepleted autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs, peptide-pulsed DCs or monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tVacDCs and sVacDCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted towards one of the three antigen-derived epitopes when priming and boosting is performed with sVacDCs. In contrast, tVac-transfected DCs prime T cells towards all antigen-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance. While CD25+ cell depletion prior to priming with sVacDCs alleviates immunodominance, cotransfection of dendritic cells with GITR-L does so in some but not all cases.

Animals↗
PubMed1961

Genetic modifiers of streptomycin resistance in Pneumococcus.

Bryan, Betty E. (University of Rochester, Rochester, N. Y.). Genetic modifiers of streptomycin resistance in pneumococcus. J. Bacteriol. 82:461-470. 1961.-Streptomycin-resistant mutants arising spontaneously in pneumococcus vary widely with respect to the maximal concentrations of streptomycin which they resist. A small random sample of these mutations tested for linkage are allelic or closely linked. One single-step mutant resisting a relatively low level of streptomycin undergoes the following changes: (i) replacement (by a secondary mutation) of the original mutation by an allele conferring a high level of resistance; (ii) reversion to complete sensitivity by back mutation at the originally mutated site; (iii) enhancement by an unlinked modifier gene which confers no resistance by itself but raises the level of resistance of the mutant strain approximately 10-fold. This modifier may have some specificity in its action on the different mutations occurring at a gene locus.The possibility is discussed that multi-step resistance may often originate by acquisition of modifiers.

Drug Resistance, Microbial↗
PubMed2005

Antibiotic resistance markers in genetically modified plants: a risk to human health?

Cotransformation with an antibiotic-resistance marker is often necessary in the process of creating a genetically modified (GM) plant. Concern has been expressed that the release of these markers in GM plants may result in an increase in the rate of antibiotic resistance in human pathogens. For such an event to occur, DNA must not be totally degraded in field conditions, and the antibiotic-resistance marker must encounter potential recipient bacteria and be taken up by them, before being integrated into the bacterial genome, and the genes then expressed. In addition, the new recombinant must overcome the physiological disadvantage of acquisition of a piece of foreign DNA, probably in conditions where the new gene does not provide a selective advantage. We review each of these stages, summarising the investigations that have followed each of these steps. We contrast the potential increase in the antibiotic resistance reservoir created by antibiotic-resistance markers in GM plants with the current situation created by medical antibiotic prescribing. We conclude that, although fragments of DNA large enough to contain an antibiotic-resistance gene may survive in the environment, the barriers to transfer, incorporation, and transmission are so substantial that any contribution to antibiotic resistance made by GM plants must be overwhelmed by the contribution made by antibiotic prescription in clinical practice.

Drug Resistance↗
PubMed2006

Screening for genetic modifiers of amyloid toxicity in yeast.

In recent years the facile, yet powerful, genetics of the baker's yeast Saccharomyces cerevisiae has been appropriated for the study of amyloid toxicity. Several models of amyloid toxicity using this simple eukaryotic organism have been developed that faithfully recapitulate many disease-relevant phenotypes. Furthermore, these models have been exploited in genetic screens that have provided insight into conserved mechanisms of amyloid toxicity and identified potential therapeutic targets for disease. In this chapter, we discuss the strengths and weaknesses of yeast models of amyloid toxicity and how experiments with these models may be relevant to amyloid disorders. We suggest approaches for development of new yeast models of amyloid toxicity and provide an overview of screening protocols for genetic modifiers of amyloid toxicity by both random and systematic approaches.

Amyloid↗
PubMedDate not supplied

Sampling grain shipments to detect genetically modified seed.

Using the binomial distribution, the effect of sample size on the variability among sample test results when sampling a lot with 1.0% genetically modified (GM) or biotech seed was evaluated. The coefficient of variation, cv, among 500-seed sample test results taken from a lot with truly 1.0% was computed to be 44.5%. Increasing sample size to 1000 seeds reduced the cv among sample test results to 31.5%. The effects of sample size and accept/reject limits on the buyer's risk (bad lots accepted) and the seller's risk (good lots rejected) was also evaluated assuming a tolerance of 1.0% GM seed. Increasing sample size decreases both the buyer's and seller's risks at the same time. Using an accept/reject limit below the regulatory tolerance decreases the buyer's risk, but increases the seller's risk. Using an accept/reject limit above the regulatory tolerance decreases the seller's risk but increases the buyer's risk.

Analysis of Variance↗
PubMed2005

Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize.

In this study, we developed a novel multiplex polymerase chain reaction (PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within a single reaction. The eight detection primer pairs designed to be construct specific for eight respective GM events (i.e., Bt11, Event176, GA21, MON810, MON863, NK603, T25, and TC1507) and a primer pair for an endogenous reference gene, ssIIb, were included in the nonaplex(9plex) PCR system, and its amplified products could be distinguished by agarose gel and capillary electrophoreses based on their different lengths. The optimal condition enabled us to reliably amplify two fragments corresponding to a construct specific sequence and a taxon specific ssIIb in each of the eight events of GM maize and all of nine fragments in a simulated GM mixture containing as little as 0.25% (w/w) each of eight events of GM maize. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM maize.

Base Sequence↗
PubMed2004

Consumer protection from an EU regulation on the mandatory labelling of genetically modified food.

On the 7. November 2003 a new regulation was enforced in the states of the EU to govern the authorisation and labelling of genetically modified food in standardized and legally binding form. Raw materials from GM crops now have to feature in the list of ingredients of the end products. The consumer is free to choose whether or not he accepts gene technology in his food purchases.

Consumer Product Safety↗
PubMed2003

The release of genetically modified crops into the environment. Part I. Overview of current status and regulations.

In the past 6 years, the global area of commercially grown, genetically modified (GM) crops has increased more than 30-fold to over 52 million hectares. The number of countries involved has more than doubled. Especially in developing countries, the GM crop area is anticipated to increase rapidly in the coming years. Despite this high adoption rate and future promises, there is a multitude of concerns about the impact of GM crops on the environment. Regulatory approaches in Europe and North America are essentially different. In the EU, it is based on the process of making GM crops; in the US, on the characteristics of the GM product. Many other countries are in the process of establishing regulation based on either system or a mixture. Despite these differences, the information required for risk assessment tends to be similar. Each risk assessment considers the possibility, probability and consequence of harm on a case-by-case basis. For GM crops, the impact of non-use should be added to this evaluation. It is important that the regulation of risk should not turn into the risk of regulation. The best and most appropriate baseline for comparison when performing risk assessment on GM crops is the impact of plants developed by traditional breeding. The latter is an integral and accepted part of agriculture.

Biotechnology↗
PubMed2005

Quantitative detection system for maize sample containing combined-trait genetically modified maize.

Various countries have established regulations that stipulate the labeling of agricultural commodities, feed, and food products that contain or are made from genetically modified (GM) material or that contain adventitious GM material in amounts that exceed certain threshold levels. While regulations in some countries refer to GM material on a weight per weight (w/w) percentage, the currently applied detection methods do not directly measure the w/w percentage of the GM material. Depending on the particular method and the sample matrix it is applied to, the conversion of analytical results to a w/w percentage is challenging or not possible. The first rapid PCR system for GM maize detection on a single kernel basis has been developed. The equipment for the grinding of individual kernels and a silica membrane-based 96-well DNA extraction kit were both significantly revised and optimized for this particular purpose, respectively. We developed a multiplex real-time PCR method for the rapid quantification of GM DNA sequences in the obtained DNA solutions. In addition, a multiplex qualitative PCR detection method allows for the simultaneous detection of different GM maize traits in each kernel and thereby for identification of individual kernels that contain a combination of two or more GM traits. Especially for grain samples that potentially contain combined-trait GM maize kernels, the proposed methods can deliver informative results in a rapid, precise, and reliable manner.

DNA, Plant↗
PubMed2001

Genetically modified feeds in animal nutrition. 2nd communication: glufosinate tolerant sugar beets (roots and silage) and maize grains for ruminants and pigs.

To analyse substantial equivalence of genetically modified sugar-beets and maize, in which the glufosinate-tolerant (Pat) gene is inserted, crude nutrients, the amino acid and the fatty acid profiles as well as the composition of the NDF-fraction of maize grains were determined and compared with those of the corresponding non-transgenic cultivars. Due to the genetic manipulation differences in crude nutrient contents including sugar and starch were not detected. The amino acid profile of maize grains was analysed to be the same. Fatty acid profile and composition of cell wall constituents did not show any influences as well. Digestibility of Pat-sugar-beets and maize grains for pigs did not demonstrate meaningful differences as compared to the corresponding non-transgenic cultivars. Digestibility of sugar-beet roots and sugar-beet top silage for ruminants proved to be also in the scope of natural variance. As the digestibility of the macro nutrients remained unaffected, the Pat-gene introduction into both crops did not show an influence on the energetic feeding value. For pigs the ME-content of Pat-sugar-beets was determined to be 14.1 MJ/kg DM versus 13.7 MJ of the non-transgenic cultivars. ME-content of Pat-maize grains was 16.0 MJ/kg DM versus 15.8 MJ for controls. For ruminants the feeding value of Pat-sugar-beets was found to be 8.5 MJ NEL/kg DM or 13.2 MJ ME/kg DM, regardless of whether the Pat-gene was inserted or not. The corresponding energy values of sugar-beet top silage ranged between 5.2 and 5.5 MJ NEL/kg DM or 8.6 and 9.1 MJ ME/kg DM, with differences considered in the biological range.

Amino Acids↗
PubMed2004

Metabolism of zearalenone by genetically modified organisms expressing the detoxification gene from Clonostachys rosea.

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45 degrees C and pH 10.5 (k(cat) at 30 degrees C, 0.51 s(-1)). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k(cat) was highest with zearalanone (1.5 s(-1)) and lowest with beta-zearalenol (0.075 s(-1)). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, beta-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuäne, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.

Escherichia coli↗
PubMed2005

Genetically modified organisms for the environment: stories of success and failure and what we have learned from them.

The expectations raised in the mid-1980s on the potential of genetic engineering for in situ remediation of environmental pollution have not been entirely fulfilled. Yet, we have learned a good deal about the expression of catabolic pathways by bacteria in their natural habitats, and how environmental conditions dictate the expression of desired catalytic activities. The many different choices between nutrients and responses to stresses form a network of transcriptional switches which, given the redundance and robustness of the regulatory circuits involved, can be neither unraveled through standard genetic analysis nor artificially programmed in a simple manner. Available data suggest that population dynamics and physiological control of catabolic gene expression prevail over any artificial attempt to engineer an optimal performance of the wanted catalytic activities. In this review, several valuable spin-offs of past research into genetically modified organisms with environmental applications are discussed, along with the impact of Systems Biology and Synthetic Biology in the future of environmental biotechnology.

Animals↗
PubMed2003

Genetic modifiers of tauopathy in Drosophila.

In Alzheimer's disease and related disorders, the microtubule-associated protein Tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles. Mutations in the tau gene cause familial frontotemporal dementia. To investigate the molecular mechanisms responsible for Tau-induced neurodegeneration, we conducted a genetic modifier screen in a Drosophila model of tauopathy. Kinases and phosphatases comprised the major class of modifiers recovered, and several candidate Tau kinases were similarly shown to enhance Tau toxicity in vivo. Despite some clinical and pathological similarities among neurodegenerative disorders, a direct comparison of modifiers between different Drosophila disease models revealed that the genetic pathways controlling Tau and polyglutamine toxicity are largely distinct. Our results demonstrate that kinases and phosphatases control Tau-induced neurodegeneration and have important implications for the development of therapies in Alzheimer's disease and related disorders.

Alzheimer Disease↗
PubMed2002

Mad cows, mad corn and mad communities: the role of socio-cultural factors in the perceived risk of genetically-modified food.

The rapid globalization of the world economy has increased the need for a knowledge base of reliable socio-cultural differences in perceptions, values and ways of thinking about new food technologies. Awareness of socio-cultural differences is important because collaborative efforts to deal with food hazards presuppose some understanding of where, how and why the viewpoints of various stakeholders may differ. In the present paper factors that influence public perceptions of genetically-modified (GM) food are discussed, with a special focus on the unique circumstances of populations in the USA, Europe and developing countries. It is argued that effective communication and decision making about the risk of GM food depends critically on understanding how socio-cultural groups differ in their values and in the way they deal with the risks and benefits of new technologies. The implications of psychological aspects of perceived risk (including the roles of qualitative dimensions of risk, world views and trust) for public acceptance of new food technologies are highlighted.

Animals↗
PubMed2006

[Laboratory-performance study of quantitative PCR methods to analyze an approved genetically modified maize (Mon810 Line)].

A laboratory-performance study was carried out to investigate factors affecting the reliability of the quantitative PCR method to analyze an approved genetically modified (GM) maize (Mon810 line). Test maize powdered samples were prepared as blind samples containing a high (assigned value; 5.45%) or low (assigned value; 0.35%) concentration of the Mon810 line. After confirmation of their homogeneity, they were provided to 27 laboratories participating in the collaborative study. The data were collected from all laboratories and statistically analyzed. Two laboratories, which used a Roche LightCycler (LC), reported significantly high test values. A further examination showed that the LC method is greatly affected by the equipment itself or PCR reagents, resulting in poor repeatability. On the other hand, some laboratories, which used ABI quantitative PCR equipment, reported erroneous test values. In these laboratories, the errors appeared to have been due to inadequate quality and/or yield of DNA. To identify factors affecting the test values, analysis of the measured values for the taxon-specific gene will be useful. Furthermore, the modified silica-gel membrane DNA extraction method made it possible to extract the required amounts of DNA more easily and in a shorter time than before.

DNA, Plant↗
PubMed2000

Predictions of biodiversity response to genetically modified herbicide-tolerant crops.

We simulated the effects of the introduction of genetically modified herbicide-tolerant (GMHT) crops on weed populations and the consequences for seed-eating birds. We predict that weed populations might be reduced to low levels or practically eradicated, depending on the exact form of management. Consequent effects on the local use of fields by birds might be severe, because such reductions represent a major loss of food resources. The regional impacts of GMHT crops are shown to depend on whether the adoption of GMHT crops by farmers covaries with current weed levels.

Agriculture↗
PubMed2002

[A detection method of recombinant DNA from genetically modified potato (NewLeaf Plus potato) and detection of NewLeaf Plus potato in snack].

A detection method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) potato (NewLeaf Plus potato; NL-P), which has not been authorized as safe in foods in Japan. The potato sucrose synthase gene was used as an internal control. The DNA from NL-P specifically provided an amplified band using PCR with a primer pair recognizing PLRV-rep gene. In addition, to prevent false-positive results in processed potato foods infected with PLRV, we designed a primer pair recognizing sequences derived from two organisms to detect specifically NL-P in processed potato. The PCR product obtained using the designed primer pair was specific for NL-P. The DNA introduced into NL-P could be detected from potato powder samples containing 0.05% NL-P. The proposed method was applied to the detection of NL-P in 25 processed potato foods. NL-P was detected in 3 snack products.

DNA, Plant↗