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October 1998 | |
NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
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October 1998 | |
NEWS FOR THE AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY COMMUNITY
IN THIS ISSUE:
FDA Addresses Use Of Antibiotic Resistance Marker genes
Greenhouse Manual In The Works - Editor Needed
Graphics Of Environmental Releases Data Now Available
The Royal Society Reports On GM Issues And Regulations
T-DNA Mediated Site-Specific Recombination
Sugar Beets Engineered To Produce Healthier Sugar
Gene Transfer Methods Compared
Cloning May Rescue Endangered Species
Engineered Rhizobia Designed For Bioremediation
Dow Ups Agbiotech Ante
The Food and Drug Administration has issued draft guidance entitled "Use of Antibiotic Resistance Marker Genes in Transgenic Plants: Guidance for Industry" and a report entitled "Report on Consultations Regarding Use of Antibiotic Resistance Marker Genes In Transgenic Plants." FDA is making both documents available for public comment. Electronic versions of the draft guidance and report are available on the Internet at http://vm.cfsan.fda.gov/~lrd/biotechm.html
FDA's 1992 policy statement regarding foods derived from genetically engineered plants specifically discussed antibiotic resistance selectable marker genes, noting that both the gene and the encoded enzyme, unless removed, are expected to be present in foods derived from plants developed using the markers. The agency acknowledged that when present in food, enzymes that inactivate certain clinically useful antibiotics theoretically might reduce the therapeutic efficacy of antibiotics administered orally.
FDA administrators maintain that it is important to evaluate such concerns with respect to commercial use of antibiotic resistance marker genes in food, and to consider the possibility that resistance to antibiotics in microorganisms has the potential to spread through horizontal transfer of such marker genes from plants. This second consideration was reflected in FDA's evaluation of the use of the kanamycin resistance gene product (neomycin phosphotransferase II, or nptII) when the agency amended the food additive regulations in 1994 to permit the use of nptII in the development of transgenic tomato, cotton, and oilseed rape.
Since that decision, the agency has continued to receive inquiries from crop developers as well as from the public regarding the safety and regulatory status of antibiotic resistance marker genes. To address the issue, a team of scientists from FDA's Center for Food Safety and Applied Nutrition, Center for Veterinary Medicine, and Center for Drug Evaluation and Research recently held consultations with outside experts having expertise in relevant fields including gene transfer and antibiotic resistance. The purpose of the consultations was to determine whether, and if so, under what circumstances, FDA should recommend that a given antibiotic resistance gene not be used in crops intended for food use. The report summarizes these consultations on the use of antibiotic resistance marker genes in transgenic plants.
The draft guidance represents the agency's current thinking on the use of antibiotic resistance marker genes in transgenic plants. It is intended to provide information to crop developers that will assist them in making decisions on the use of these genes in the development of transgenic plants. The following is an excerpt from the draft guidance.
"FDA acknowledges that the likelihood of transfer of an antibiotic resistance marker from plants to microorganisms in the gut or in the environment is remote and that such transfer, if any, would likely be insignificant when compared to transfer between microorganisms, and in most cases, would not add to existing levels of resistance in bacterial populations in any meaningful way. Nonetheless, FDA believes that developers should evaluate the use of antibiotic resistance marker genes in crops on a case-by-case basis taking into account information on 1) whether the antibiotic is an important medication, 2) whether it is frequently used, 3) whether it is orally administered, 4) whether it is unique, 5) whether there would be selective pressure for transformation to take place, and 6) the level of resistance to the antibiotic present in bacterial populations. If a careful evaluation of the data and information suggests that the presence of the marker gene or gene product in food or feed could compromise the use of the relevant antibiotic(s), the marker gene or gene product should not be present in the finished food or feed. FDA notes that certain antibiotics are the only drug available to treat certain clinical conditions (e.g., vancomycin for use in treating certain staphylococcal infections). Marker genes that encode resistance to such antibiotics should not be used in transgenic plants."
In announcing the availability of the report and draft guidance, FDA acknowledges that there may be relevant issues not addressed, e.g., the likelihood of a mutation in a given antibiotic resistance gene giving rise to resistance to another antibiotic. The agency encourages comments on issues that may have not been covered.
Written comments (two copies) on the draft guidance and report should be submitted by December 7, 1998 to the Dockets Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. Comments should be identified with the Docket No. 98D-0340. After consideration of any comments received, FDA will revise the draft guidance as appropriate and announce its availability in the Federal Register.
How many greenhouses in the U.S. have transgenic plants growing in them? Certainly more than a hundred, maybe quite a few more. How many greenhouse managers know what conditions provide appropriate levels of containment for genetically engineered plants and microbes? The answer here is probably a much smaller number. An informal survey earlier this year indicated that researchers and greenhouse managers, particularly at academic institutions, are often unsure about biosafety requirements under Appendix P of the NIH Guidelines (January 1996) and how to meet them. As a result, their experimental procedures may be excessively restrictive, unwittingly lax, or somewhere in between.
Information Systems for Biotechnology has begun a project to develop a manual on "Greenhouse Research with Transgenic Plants and Microbes: A Common Sense Guide to Containment." The manual is intended to provide researchers and greenhouse managers with a practical guide to greenhouse design and procedures for conducting experiments under appropriate conditions. The goal is to address physical and biological approaches to containment, emphasizing what is reasonable and prudent.
ISB is looking for a collaborator to manage the project now that preliminary information has been collected from academic and industry sources. The position entails gathering additional information, identifying people to write chapters, planning and coordinating graphics and illustrations, editing the text, and assembling the final document into a practical reference. The manual will be published on the Internet and distributed in print copy.
Interested individuals familiar with transgenic greenhouse design and/or operations and having excellent writing and organizational skills are encouraged to contact Pat Traynor for more information (phone: 540-231-2620; email: traynor@nbiap.biochem.vt.edu).
Charts and maps that summarize key information from the Environmental Releases databases have been added to the ISB website. The graphics complement ISB's web-based program for searching the complete set of field test and deregulatory petition records from USDA/APHIS. The graphics are automatically generated each time ISB receives an update from USDA/APHIS, and thus present the most current data available.
Pie charts, bar charts, and U.S. maps illustrate at a glance key information including:
Examples of these charts follow. The actual charts can be found on the ISB website (http://www.isb.vt.edu); select Databases, then Field Tests, then click on "Go To Charts." They are provided as .gif files that can be downloaded and incorporated into presentation materials. Please include attribution to Information Systems for Biotechnology.
(Map shown at 75% size)
The latest development in the ongoing European debate concerning genetically modified (GM) crops is a report from a special working group of the Royal Society, a highly respected and influential scientific organization in the UK. The report entitled "Genetically Modified Plants for Food Use" is the product of a number of prominent experts in a variety of fields. It concisely reviews current regulatory controls both in the UK and in Europe, and addresses questions of gene transfer from GM crops, other specific concerns of environmental harm, and issues related to GM plants as food. Possible future developments are also discussed with suggested revisions to the current regulatory system.
One of the key proposals put forward by the working group is for an "over-arching body to monitor the impact of GM crops on agronomic practices or to look at the cumulative effects of such crops." At present, applications to field test or market GM crops are reviewed case by case and by a host of special committees with very specific mandates. The report suggests that each of the individual committees (such as the Food Advisory Committee, the Advisory Committee on Releases into the Environment, the Advisory Committee on Novel Foods and Processes, and the Advisory Committee on Genetic Modification) cannot consider many of the wider implications of genetic technology for practical reasons. Some of the tasks which the proposed "super regulator" might undertake are:
In the face of increased calls for a moratorium on field trials of GM crops in the UK by groups such as the Friends of the Earth, the Royal Society report states that "many of the concerns raised cannot be addressed without the information gained from long-term small-scale field trials and laboratory work." Hence a moratorium is not considered the most appropriate way ahead.
Throughout the report attention is drawn to the argument that the risks often associated with GM crops are the same risks that have been taken with conventional agricultural techniques. For example, traditional plant breeding methods have been used to develop herbicide resistant crops. This practice has been ongoing for some time and there has been no observed increase in the incidence of herbicide tolerant weed species. There is therefore no reason to expect that we would see an increase as a result of cultivating GM herbicide resistant crops. The problem of weed control is considered as a general agricultural issue that requires effective management, crop rotation and the use of selective herbicides. However, the proliferation of GM herbicide resistant crops might be a problem if farmers do not adhere to crop management guidelines.
The report recognizes that it is inevitable that genetically modified material will enter all of our diets, even if at very small levels. This is a consequence of (a) the possibility of cross-pollination in our fields from GM crops to non-GM crops, and (b) the difficulty of testing foodstuffs for GM material.
Cross-pollination has become an issue of importance for organic farmers who fear that their "organic" status will be revoked if they cannot guarantee their produce to contain no modified genetic material. The Royal Society report suggests that minimum acceptable levels will have to be introduced by the licensing bodies. This will not please the organic farming community.
Similarly, food retailers will have to accept that for the purposes of labeling GM foods as such, enforcement will only be possible if it is recognized that there is a "minimum level for adventitious presence of GM material, below which a product can be considered to be free of GM derivatives." This will not please the consumer lobby who have already suffered a set-back with the recent European Novel Food Regulation relating to food labeling. The regulation has been predicated on the existence of adequate testing technology which simply does not exist at present. Effectively, if we cannot detect a difference, or do not have a test to detect a difference, then we must treat GM and non-GM foods in the same way in the law.
Overall the report covers many of the key areas under debate: gene transfer from GM plants, antibiotic resistance, insect tolerant crops, herbicide tolerant crops, virus resistant crops, labeling and GM/non-GM crop segregation, allergenic risks in GM foods, etc. The science is up to date and there are useful appendices setting out the membership of the Government advisory committees on GM plant technology. There will be little to please the hard-line environmentalists and consumerists, but the suggestion of an over-arching regulatory committee does seem to be a significant proposal.
The report text is available in full at: http://www.royalsoc.ac.uk/st_pol40.htm. Copies may be obtained free of charge from The Science Advice Section at the Royal Society (email: angela.halpin@royalsoc.ac.uk).
Ben Thomson
Department of Law
University of Sheffield
lwp97bt@sheffield.ac.uk
In mammalian cells, the introduction of DNA at specific chromosomal sites via homologous recombination has successfully been applied to inactivate or modify specific genes. Such gene targeting can be done in plants as well, but has not been widely attempted due to the low efficiency with which transfected DNA recombines with homologous sequences in the plant genome. Use of site-specific recombination systems may have great potential for overcoming this limitation.
In a study carried out at the Institute of Molecular Plant Sciences at Leiden University in the Netherlands, the bacteriophage P1 Cre/lox system was used to insert Agrobacterium tumefaciens transferred DNA (T-DNA) into a specific chromosomal location in Arabidopsis thaliana. The Cre/lox system, consisting of a recombinase protein (Cre) and short 34 base pair recombination sites (lox), catalyses precise and stable insertional recombination between the respective DNA target sites.
The Leiden group first genetically engineered Arabidopsis target lines which harbored a single chimeric insert comprised of an active promoter, a lox sequence, and a Cre gene. Root explants from transgenic seedlings were co-cultivated with an Agrobacterium strain harboring a T-DNA vector carrying a promoterless lox-neomycin phosphotransferase (nptII) fusion. Cre-mediated site-specific recombination of the T-DNA at the genomic lox site restored nptII expression by translational fusion to the promoter at the target locus. Simultaneously, the recombinase gene was disconnected from the promoter by displacement, thereby preventing the efficient reversible excision reaction. Recombinants could be selected on the basis of kanamycin resistance.
Of the site-specific recombinants, 89% were the result of precise integration. T-DNA, which is transferred to plant cells as a single-stranded linear DNA structure, is in principle incompatible with Cre-mediated integration, which requires circular double stranded DNA. Nevertheless, this study clearly demonstrates the feasibility of Agrobacterium mediated transformation to obtain site-specific integration. This approach may lead to development of more efficient systems, applicable to many plant species, that could target a transgene to a single pre-selected chromosomal site, thus eliminating variation in gene expression level.
Source
Vergunst, A.C., L.E.T. Jansen, and P.J.J. Hooykaas. 1998. Site specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre-recombinase. Nucleic Acids Research 26:2729-2734.
P. Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise
Hyderabad, India
ipe@hyd.ap.nic.in
SUGAR BEETS ENGINEERED TO PRODUCE HEALTHIER SUGAR
Dutch scientists have developed a new sugar beet that produces fructan, a low calorie sweetener and a healthier alternative to sucrose. Reporting in the September issue of Nature Biotechnology, researchers at the Center for Plant Breeding and Reproduction Research at Wageningen describe how they developed "fructan beets" by inserting a single gene from Jerusalem artichoke that encodes an enzyme for converting sucrose to fructan (1). The new beet has the potential to replace more expensive fermentation methods of producing low molecular weight fructans, which are nearly as sweet as regular sugar but indigestible by humans.
The preferences of health-conscious consumers are increasingly shifting away from high-calorie sucrose. Fructans, first described by a German scientist in 1804, are polymers of fructose having considerable appeal to the food industry as they are not metabolized by humans and have no caloric value. Further, their chemistry makes them ideal substitutes for fat and thus suitable for low-calorie bakery and dairy products (2). Fructans, like yogurt, also promote the growth of beneficial bacteria present in the gut. In fact, swine and poultry feeds are often supplemented with fructans to reduce the smell of the animals' waste. Fructans are also implicated in reducing the risk of human diseases such as insulin-dependent diabetes and obesity, and may play a role in reducing blood cholesterol levels.
While fructans such as inulin are found naturally in many plants such as chicory, their low yield and problems with enzyme degradation during extraction have minimized their commercial application. Fructans produced industrially using Aspergillus enzymes in bioreactors are prohibitively expensive. Earlier studies in tobacco and potato have shown that plant metabolic pathways can be altered to produce high molecular weight fructans.
The Dutch group, led by Andries Koops, focused on sugar beet, a prodigious producer of carbohydrates under temperate conditions. Koops says that "this study shows that introduction of a carefully selected gene in the right crop and the right tissue can induce that crop to produce a new product with new market perspectives. This also shows the validity of the concept 'the plant as a biochemicals factory', a concept that already is being exploited for other plant-product combinations, although from now on the emphasis will be more on products for non-food applications."
As sugar beet was initially recalcitrant to genetic transformation, a novel method to insert genes via stomatal guard cell protoplasts was developed. Scientists then cloned the gene encoding 1-sucrose:sucrose fructosyl transferase (1-sst), the enzyme that converts sucrose into low molecular weight fructans, and introduced it into sugar beet. The tap root of transformed plants was chock full of low molecular weight fructans. While the transgenic roots produced the same amount of total sugar as the control lines, expression of the 1-sst gene resulted in conversion of more than 90% of the stored sucrose into fructans. Leaves of transgenic plants showed negligible amounts of the polymer, and control plants showed no detectable fructan. Under greenhouse conditions, the fructan beets looked normal and had nearly the same amount of root dry weight as normal sugar beet.
If field trials validate these results, the new beet has great farm potential as an inexpensive source of a versatile, nondigestible sweetener with health promoting properties. That in itself is promising, but there is more to the story. T.J. Higgins, CSIRO, Australia, comments: "This is a dramatic demonstration of the power of metabolic engineering using transgenic plants. As well as describing a potentially interesting method for the production of low molecular weight fructans from sucrose, the authors have produced plants useful for the study of carbon partitioning in leaves and modified roots."
In addition, fructan accumulation in plants has been shown in many studies to be linked to drought and cold tolerance, so the new beets may turn out to be hardier as well. Further, as fructans have potential industrial applications such as the manufacture of biodegradable plastics and fabric softeners, fructan beets may also end up in farm factories to produce industrial feed stocks.
Sources
1. Sevenier, R., R.D. Hall, I. van der Meer, H.J.C. Hakkert, A.J. van Tunen, and A.J. Koops. 1998. High level fructan accumulation in a transgenic sugar beet. Nature Biotechnology 16:843-846.
2. Biotechnology puts sugar at heart of healthy diet. http://www.mpiz-koeln.mpg.de/~amica/focus/topics97/0301/0301.htm
C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@tusk.edu
GENE TRANSFER METHODS COMPARED
When transformation was first achieved by exploiting the natural ability of Agrobacterium tumefaciens to insert foreign genes into plants, it was envisaged that this approach would be the ultimate plant gene transfer procedure. It soon became apparent, though, that the method was not equally straightforward and efficient for all crop species.
Consequently, alternative transformation techniques for direct gene transfer into plant tissues have been developed. These include micro- and macroinjection, tissue electroporation and electrophoresis, particle bombardment, use of silicon carbide fibers and laser microbeams, and direct DNA uptake into isolated protoplasts.
A group of scientists in the Plant Research Group at the University of Nottingham (UK) recently evaluated four methods for direct gene transfer into intact tissues of maize. The techniques compared were particle bombardment, tissue electroporation, silicon carbide-mediated gene transfer, and tissue electrophoresis.
The techniques were evaluated for their efficacy in transforming immature embryos and Type II callus of A188 maize, by measuring transient expression of the GUS reporter gene. High levels of GUS expression were observed in callus subjected to either tissue electroporation or particle bombardment. In immature embryos, only particle bombardment resulted in high GUS activity. Very low levels of expression were achieved with silicon carbide-mediated gene transfer. GUS activity was not obtained following tissue electrophoretic gene delivery.
In addition to transformation frequency, other factors must be taken into account when considering different methods. Among these are ease of use, rapidity of processing samples, amount of plasmid DNA required per sample, and cost. Although particle bombardment is a simple and efficient technique, the standard apparatus used for this is expensive. However, there are alternative designs using different propelling forces which can be constructed at a relatively low cost. In contrast, silicon carbide and electrophoresis treatments have very low start-up costs but, along with tissue electroporation, require large aliquots of plasmid DNA per sample.
The results in this study for transient gene expression obtained with tissue electroporation and particle bombardment compare favorably with earlier reports. However, the efficiencies of tissue electrophoresis and silicon carbide-mediated gene transfer are considerably lower in this study. Although the simple silicon carbide approach may have potential as a method to generate transgenic plants in the future, considerable experimentation is still required for this technique to be a realistic approach when initiating transformation programs with new crops.
Source
Southgate, E.M., M.R. Davey, J.B. Power, and R.S. Westcott. 1998. A comparison of methods for direct gene transfer into maize (Zea mays). In vitro Cell. Dev. Biol. Plant 34:318-224.
P. Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise
Hyderabad, India
ipe@hyd.ap.nic.in
CLONING MAY RESCUE ENDANGERED SPECIES
Lost among the sensational scenarios of human cloning were the practical applications of animal cloning to agriculture. One of the many proposed uses for cloning by nuclear transfer was the preservation of endangered species. Now scientists in the Ruakura Research Centre in New Zealand have reported the use of cloning to preserve a vanishing breed of cattle.
The last remaining survivor of the Enderby Island breed is a seven year old female. This cow, named Lady, has been living in isolation on a cold-climate island near New Zealand. Repeated attempts to propagate the breed by artificial insemination have resulted in only a single male calf.
Using somatic cell nuclear transfer technology, which was first successfully demonstrated in sheep and then replicated in cows and mice, a female clone of Lady has been produced. The calf, named Elsie, is now a few weeks old. The plan, once she becomes reproductively mature, is to inseminate Elsie with semen that was previously obtained from culled males and has been stored frozen. Semen from the one male calf produced by Lady could be used, but this could lead to genetic problems due to inbreeding. Thus use of the frozen semen would be preferable in order to maintain genetic diversity.
As species become endangered due to reduction of sustainable breeding populations, cloning by nuclear transfer provides a possible means of saving those species with unique characteristics. The Enderby Island breed has adapted to sub-Arctic conditions and thus contains a genetic makeup which confers cold-tolerance. These genes may be valuable for increasing cold-tolerance of other cattle breeds.
Eric Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu
ENGINEERED RHIZOBIA DESIGNED FOR BIOREMEDIATION
The broad-host-range plasmid RP4::TOL has been transferred from Escherichia coli to a Rhizobium meliloti strain by researchers at Howard University. RP4::TOL carries the benzene, toluene, and xylene degradative gene cluster. These toluene-based compounds include fuels, solvents and similar hydrocarbons which become contaminants when in the soil. The project was funded primarily by the U.S. Army and involved cooperation with researchers at the USDA/ARS lab in Beltsville, Maryland.
Rhizobium meliloti is the nitrogen fixing microsymbiont of alfalfa. The advantage of using a legume symbiont as a bioaugmentation agent is twofold. First, the legume host will feed and provide a home for the bacteria in the soil; second, the alfalfa legume is deeply rooted which hopefully will carry the rhizobia deep into the contaminated soil.
A paper on the initial stages of this work was presented at the North American Conference on Symbiotic Nitrogen Fixation in February of this year. An abstract of the continuing work has been submitted to the coming international bioremediation meeting.
Howard University has applied for a patent. However, lead researcher Dr. Sisur Dutta indicated that it might be some time before the strain is ready for practical use. Bioremediation experiments still need to be done under field conditions, not just in the greenhouse as has been the case to date; and the EPA must approve the safety of the organism before commercial release. Further complicating Dr Dutta's strategy and plans is the fact that the U.S. Army has cut much of the funding for the project.
Tom Wacek
Urbana Laboratories
twacek@seedsolutions.com
The latest move by a major agricultural chemical company to bolster its position in the biotech battle field is the formation of Advanced Agritraits LLC by Dow Agrosciences LLC, a subsidiary of Dow Chemical. The company is being formed to act as a clearinghouse for companies looking for a more effective way of commercializing their technologies. Advanced Agritraits will do this through strategic alliances, joint ventures, acquisitions, and/or licensing agreements (1).
The company will promote relationships with partners interested in offering or acquiring access to technology, including access to germplasm with preferred traits such as insect or disease resistance, herbicide tolerance, oil content modification, and enhanced nutritional qualities. Dow Agrosciences will initiate this process by contributing access to its own proprietary genes and a variety of crop traits, including certain insect-related technology.
Advanced Agritraits is seeking to provide companies with more risk-averse, cost-effective, and efficient mechanisms to build on their own technology strengths. Their approach is to develop "cassettes" of desirable traits which can be placed into the germplasm of a variety of different plants. The concept is to use multiple cassettes to develop plants with a set of desirable traits for very specific conditions. Advanced Agritraits will make these cassettes available through non-exclusive licenses to interested parties such as seed companies, who can select from the variety of available traits (2).
As evidence of the interest in this new business model, Illinois Foundation Seeds, Inc. (IFSI) has acknowledged its intent to license to Advanced Agritraits rights to a substantial portion of its portfolio of proprietary germplasm. Through the agreement, which is proposed to involve some level of exchange of ownership interests, IFSI will receive non-exclusive marketing rights to the new trait-enhanced products, among other consideration. One of the drivers of the deal for IFSI is the ability to provide its customers with high quality corn genetics combined with superior value-enhanced trait technology. IFSI is the second largest foundation seed company in the U.S. (1).
Dow also announced the formation of an alliance with functional genomics company Biosource Technologies. Biosource, which has developed a mechanism to express genes from plants and assay gene function, will collaborate with Dow Agrosciences to create specific traits. Dow will receive rights to all genes that relate to useful crop traits. Biosource maintains rights to all pharmaceutical and animal health applications (2).
The formation of the new venture and strategic alliances is part of Dow Chemicals efforts to take part in the industry-wide shift toward biotechnology as a instrumental driver of future earnings.
Sources
1. Dow AgroSciences Forms New Biotechnology Company. News Release, September 8, 1998, http://www.dowagro.com/main/company/news/news026.htm
2. Seachrist, L. Dow Forms New Company For Agbiotech Partnering. BioWorld Today, Sept. 10, 1998, Vol. 9, No. 174, pp. 1-4.
William O. Bullock
Institute for Biotechnology Information
Research Triangle Park, NC
wbullock@mindspring.com
The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. It does not necessarily reflect the views of the U.S. Department of Agriculture or of Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge. P.L. Traynor, Editor.
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