A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.

SummaryIn 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.

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A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately threequarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed threedimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype. INTRODUCTIONOver the past two decades, Arabidopsis has become a model organism for research in plant physiology, development, biochemistry, and pathogenesis. Recent developments in Arabidopsis genomics research include the construction of a nearly complete physical map of the genome based on yeast artificial chromosomes (Choi et al., 1995;Schmidt et al., 1996Schmidt et al., , 1997Zachgo et al., 1996;Camilleri et al., 1998), bacterial artificial chromosomes (BACs; Mozo et al., 1998), P1 clones (Liu et al., 1995Kaneko et al., 1998), the systematic sequencing of expressed sequence tags (ESTs; Höfte et al., 1993;Newman et al., 1994), and coordinated multinational sequencing efforts (Goodman et al., 1995;Bevan et al., 1998).The large amount of sequence information generated by Arabidopsis genome and EST sequencing projects will provide the basis for systematic studies of gene function and eventually will allow for unlimited comparisons with the yeast (Mewes et al., 1997) and Caenorhabditis ( C. elegans Sequencing Consortium, 1998) genomes. Such cross-kingdom analyses might reveal those genes that are necessary for the development and maintenance of eukaryotic cells and those that are associated with functions unique to either plants or animals. An intriguing finding to have emerged already from these studies is that half of the Arabidopsis ESTs bear no significant similarity to sequences from the previously established databases (Rounsley et al., 1996).Over the past decade, insertional mutagenesis has proven to be one of the most efficient ways of isolating and identifying genes via the traditional approaches of "forward" genetics (Feldmann, 1991;Koncz et al., 1992;Aarts et al., 1993;Jones et al., 1994;Okuley et al., 1994;Azpiroz-Leehan and Feldmann, 1997). In light of the ongoing accumulation of ever more DNA sequences of unknown function, insertional mutagenesis now offers the means for determining the function of newly elaborated sequences by a process of "reverse" genetics. The strategy begins with the broad insertional mutagenesis of the genome through the use of T-DNA or transposons followed by the identification of...

A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately threequarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed threedimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype. INTRODUCTIONOver the past two decades, Arabidopsis has become a model organism for research in plant physiology, development, biochemistry, and pathogenesis. Recent developments in Arabidopsis genomics research include the construction of a nearly complete physical map of the genome based on yeast artificial chromosomes (Choi et al., 1995;Schmidt et al., 1996Schmidt et al., , 1997 Zachgo et al., 1996;Camilleri et al., 1998), bacterial artificial chromosomes (BACs; Mozo et al., 1998), P1 clones (Liu et al., 1995Kaneko et al., 1998), the systematic sequencing of expressed sequence tags (ESTs; Höfte et al., 1993;Newman et al., 1994), and coordinated multinational sequencing efforts (Goodman et al., 1995;Bevan et al., 1998).The large amount of sequence information generated by Arabidopsis genome and EST sequencing projects will provide the basis for systematic studies of gene function and eventually will allow for unlimited comparisons with the yeast (Mewes et al., 1997) and Caenorhabditis ( C. elegans Sequencing Consortium, 1998) genomes. Such cross-kingdom analyses might reveal those genes that are necessary for the development and maintenance of eukaryotic cells and those that are associated with functions unique to either plants or animals. An intriguing finding to have emerged already from these studies is that half of the Arabidopsis ESTs bear no significant similarity to sequences from the previously established databases (Rounsley et al., 1996).Over the past decade, insertional mutagenesis has proven to be one of the most efficient ways of isolating and identifying genes via the traditional approaches of "forward" genetics (Feldmann, 1991;Koncz et al., 1992;Aarts et al., 1993;Jones et al., 1994;Okuley et al., 1994;Azpiroz-Leehan and Feldmann, 1997). In light of the ongoing accumulation of ever more DNA sequences of unknown function, insertional mutagenesis now offers the means for determining the function of newly elaborated sequences by a process of "reverse" genetics. The strategy begins with the broad insertional mutagenesis of the genome through the use of T-DNA or transposons followed by the identification o...