Can a participatory approach contribute to food chain risk analysis?

We consider food chain risks and specifically address stakeholder participation in the risk analysis process. We combine social and natural science perspectives to explore the participation process in relation to food risks and, in particular, to consider how some specific participation processes might be scientifically evaluated and how stakeholder participation in general might be incorporated into food risk decision making. We have built considerations based on three large integrative case studies that examine aspects of participatory processes. Here we use the case studies collectively to illustrate observations and beliefs concerning the nature of the interaction of stakeholders with established quantitative risk methodologies. This account is not supported by any large volume of analysis. The views in the report are expressed in relation to an accepted risk analysis framework and also with respect to probabilistic modeling of risks and are illustrated where possible with anecdotal reports of actual case study events.

Immune responses, not promoter inactivation, are responsible for decreased long-term expression following plasmid gene transfer into skeletal muscle.

Long-term high-level in vivo gene expression appears to depend on the promoter chosen to drive the gene of choice. In many cases the promoter appears to 'switch off' some time after in vivo gene transfer. We demonstrate that, following intramuscular injection of beta-galactosidase reporter plasmids, promoter 'switch off' is due to elimination of fibres expressing the transferred reporter gene by activation of a Th1 (cytotoxic) immune response. This finding, in the absence of stimulation of the immune system by viral vector proteins, has implications not only for gene transfer experiments but for the future of muscle-directed gene therapy.

Novel transcribed sequences neighbouring a translocation breakpoint associated with schizophrenia.

A 1.3Mb chromosome 11-specific yeast artificial chromosome (YAC) that spans a t(1;11) translocation breakpoint associated with major psychosis has been used to enrich cDNAs that are encoded within it and expressed in the human foetal brain. Database analysis of the selected fragments led to the identification of 54 clones matching alpha-tubulin, 4 fragments matching two anonymous human expressed sequence tags (ESTs) and 8 fragments giving no database matches. The clones matching alpha-tubulin led to the identification of a novel alpha-tubulin locus located approximately 250 kb proximal to the translocation breakpoint. Extensive sequence and expression analysis of this locus suggests that this is a processed pseudogene, although a long open reading frame is maintained and the possibility that an abnormally acting protein may be expressed in a highly tissue or developmental specific manner cannot be discounted. The novel cDNA fragments map up to 700 kb proximal to the translocation breakpoint and are associated with potential CpG islands. Reverse transcriptase polymerase chain reaction (RT-PCR) expression analysis and high resolution genomic mapping suggest that they may comprise up to three novel genes. No major disruption of the identified fragments could be detected in the genomic DNA of translocation carriers. The psychosis associated with this translocation may therefore be due to position effects on the transcription of these genes or an involvement of translocated chromosome 1 sequences.

A long-range restriction map across 3 Mb of the chromosome 11 breakpoint region of a translocation linked to schizophrenia: localization of the breakpoint and the search for neighbouring genes.

A balanced t(1;11)(q42.1;q14.3) translocation segregates with schizophrenia and related mental illness in a single large Scottish pedigree. We have constructed a long-range restriction map covering at least 3 Mb of the chromosome 11 breakpoint region and conducted searches for genes whose expression could be altered by the translocation, resulting in schizophrenia. Novel transcribed sequences of unknown function clustered around putative CpG islands, located approximately 500 kb and 700 kb above the breakpoint, represent the only evidence to date for expressed genes within the mapped region.

Pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was originally developed as a technique for providing electrophoretic karyotypes of micro-organisms. Since then the technique has evolved and diversified in many new directions. This review traces the evolution of PFGE, summarizes our understanding of its theoretical basis, and provides a comprehensive description of the methodology. Established and novel applications are explored and the reader is provided with an extensive list of references.

Evaluation of plasmid DNA for in vivo gene therapy: factors affecting the number of transfected fibers.

Gene transfer by intramuscular injection of plasmid DNA has potential application in gene therapy. We examined factors affecting the number of expressing fibers, in contrast to total expression, following injection of plasmid DNA. Barium chloride proved effective in inducing muscle necrosis and regeneration in mice, and this increased the number of fibers expressing a reporter gene. Coinjection of ion-channel modulators did not increase the number of positive fibers, but increasing dose and repeated administration of plasmid did. Importantly, the plasmid size (7-16 kb) did not affect the number of fibers expressing the transgene, in both normal and regenerating muscle.

Investigations of the F conjugation gene traI:traI mutants and lambdatraI transducing phages.

A series of traI point and deletion mutants of Flac, and a traM mutant, were characterised. Complementation tests with an amber Flac traI mutant confirmed their genotypes, and in addition all the traI mutants, but not the traM mutant, were complemented by pRS31 (PSC101 traDI) and EDlambda109 (lambdatraI). Judging from the efficiencies of plating of F-specific phages, none of the mutations affected pilus formation. The traI products of F and of the F-like plasmid R1 were interchangeable with each other but not with that of R100, while the traM product of F could not be replaced by those of R1 or of R100. Neither traI nor traM were needed for conjugal transfer of ColE1. Three lambda transducing phages carrying traI were isolated by in vivo or in vitro techniques, and characterised by genetic complementation tests, by analysis of the fragments produced by restriction endonucleases, and by measurement of heteroduplex molecules. The genetic structures together with the sizes and F coordinates, of the transfer regions carried by the phages were thereby determined. Comparison of the proteins synthesised in UV-irradiated cells by one of the lambdatraI phages with those made by a derivative carrying an amber traI mutation, allowed the traI product to be identified as a protein of molecular weight 174,000. In addition, the molecular weights of the traD (84,000), traS (18,000), and traT (25,000) products made by the lambdatraSTD1 phage EDlambda107 were measured. The possible roles of the traI and traM products in conjugation are discussed.

Characterisation of a lambda transducing phage carrying the F conjugation gene traG.

A lambda transducing phage carrying the traGSTD genes of the E. coli K12 factor F was isolated by an in vivo technique, and characterized in tra complementation tests, by determining its restriction endonuclease fragment sizes, and by measuring heteroduplex molecules. The size and location on the F physical map of the tra transducing segment was thereby determined. Comparison of the proteins synthesized in UV-irradiated cells by this phage and by a derivative carrying the amber traG79 mutation, allowed the traG product to be identified as a protein of molecular weight 100,000. In the same experiments, the sizes of the traT and traD products made by the phage were also measured, being 25,000 and 85,000 daltons respectively.

Semiconductor-controlled contour-clamped homogeneous electric field apparatus.

The design and construction of a transistor-driven hexagonal contour-clamped homogeneous electric field (CHEF) apparatus is discussed in detail. The addition of computer control of pulsed-field timings and experiment duration gives rise to an efficient electrophoresis tool designed to achieve separation of DNA molecules in different size groupings. In particular, pulse time regimes which lead to the monotonic separation of DNA molecules ranging from 90 kbp to over a megabase pair are demonstrated. Theoretical treatment of electric field clamping with transistor-driven multiple electrodes is supported by measurements and by the actual performance of electrophoretic separation of yeast chromosomes. The large sample capacity of gels run in this apparatus coupled with the modest power requirements necessary to provide a homogeneous electric field offer significant advantages over earlier CHEF designs.

A cluster of transfer RNA genes (TRM1, TRR3, and TRAN) on the short arm of human chromosome 6.

We have isolated two lambda clones that contain three transfer RNA (tRNA) genes (TRM1, TRR3, and TRAN). Both clones map to the same region (6p21.2-p22.3) of the short arm of chromosome 6. One clone contains a methionine tRNA gene and also an arginine tRNA gene, the first such human gene to be described. The other clone contains an alanine tRNA gene, again the first such human gene to be reported, and it differs from the species of human alanine tRNA transcripts sequenced to date. These clones have been used to investigate the structure of this tRNA gene cluster. The results of both conventional and pulsed-field gel analysis suggest that the alanine tRNA gene is a member of a low-copy repeat series at this location. The other clone is not located within this domain and appears to be a unique segment of DNA. Nevertheless, we also show that at least half of the methionine tRNA genes are located on the short arm of this chromosome, and if these are also located at 6p21.2-p22.3, this would constitute another major tRNA locus in human.

Long-range structure of H-ras 1-selected transgenomes.

We have used chromosome-mediated gene transfer (CMGT) and whole cell fusion to derive human-mouse hybrid cells carrying reduced human chromosomes 11, by selecting for expression of the transforming H-ras 1 oncogene. To realize the full potential of these somatic cell genetic techniques as resources for enriched DNA probe isolation and the fine structure mapping of chromosomes, the nature of any molecular rearrangements that may accompany the process of DNA transfer must be understood. We have analyzed the long-range structure of our transgenomes by pulsed field gel electrophoresis (PFGE) and show here that, whereas during cell fusion several megabase pairs (Mb) of DNA can be transferred intact, multiple rearrangements of DNA accompany CMGT even in transgenomes where other methods of analysis gave no indication of such molecular scrambling.

Extensive telomere repeat arrays in mouse are hypervariable.

In this study we have analysed mouse telomeres by Pulsed Field Gel Electrophoresis (PFGE). A number of specific restriction fragments hybridising to a (TTA-GGG)4 probe in the size range 50-150kb can be detected. These fragments are devoid of sites for most restriction enzymes suggesting that they comprise simple repeats; we argue that most of these are likely to be (TTAGGG)n. Each discrete fragment corresponds to the telomere of an individual chromosome and segregates as a Mendelian character. However, new size variants are being generated in the germ line at very high rates such that inbred mice are heterozygous at all telomeres analysable. In addition we show that specific small (approximately 4-12kb) fragments can be cleaved within some terminal arrays by the restriction enzyme MnII which recognises 5'(N7)GAGG3'. Like the complete telomere-repeat arrays (TRA's) these fragments form new variants at high rates and possibly by the same process. We speculate on the mechanisms that may be involved.

A fission yeast chromosome can replicate autonomously in mouse cells.

To test the functional capacity of a fission yeast chromosome in mouse cells, a strain of the fission yeast Schizosaccharomyces pombe, ED628 Int5, was constructed. A plasmid bearing the SV2NEO gene, which can confer G418 resistance to mouse cells, was integrated at the ura4 locus on S. pombe chromosome III. S. pombe Int5 chromosomes were introduced into mouse C127 cells by PEG-facilitated protoplast fusion. Here we describe two independent G418-resistant cell lines with distinct growth characteristics, F1.1 and F7.1, and examine the structure of material derived from S. pombe Int5 chromosome III in these lines. F1.1 is shown to contain a single rearranged block of chromatin from S. pombe chromosome III integrated into a mouse chromosome, maintained in the absence of selection. In contrast, the data for F7.1 are consistent with the presence of linear, unintegrated copies of S. pombe chromosome III, which are apparently intact and maintained in an unstable but autonomous state. The unstable maintenance of this chromosome may be due to defective centromere function leading to missegregation at mitosis or to over- or underreplication.

Human repeat-mediated integration of selectable markers into somatic cell hybrids.

We describe a strategy to introduce preferentially the dominant selectable marker neoR into the human chromosome within a monochromosome hybrid cell line. Integration of a construct containing the marker is mediated by human-specific repeat elements that promote multilocus human-specific integration with a single targeting vector. We tested two classes of repeat elements: the Alu family of SINE repeats and the Line1 repeat family. We show that Alu sequences alone are insufficient to direct human-specific integration but when used in combination with a Line1 element, or when only Line1 elements are included, integration of the vector into the human component of a monochromosome somatic cell hybrid is favored. The vectors also carry sequences that facilitate mapping and selective cloning of the targeted region. This strategy provides a means to generate selectable human subchromosomal fragments that can be used for localization of genes through positional cloning and, more important, for the identification of functional units through DNA transfer.