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.

A 6.9-Mb high-resolution BAC/PAC contig of human 4p15.3-p16.1, a candidate region for bipolar affective disorder.

Bipolar affective disorder (BPAD) is a complex disease with a significant genetic component and a population lifetime risk of 1%. Our previous work identified a region of human chromosome 4p that showed significant linkage to BPAD in a large pedigree. Here, we report the construction of an accurate, high-resolution physical map of 6.9 Mb of human chromosome 4p15.3-p16.1, which includes an 11-cM (5.8 Mb) critical region for BPAD. The map consists of 460 PAC and BAC clones ordered by a combination of STS content analysis and restriction fragment fingerprinting, with a single approximately 300-kb gap remaining. A total of 289 new and existing markers from a wide range of sources have been localized on the contig, giving an average marker resolution of 1 marker/23 kb. The STSs include 57 ESTs, 9 of which represent known genes. This contig is an essential preliminary to the identification of candidate genes that predispose to bipolar affective disorder, to the completion of the sequence of the region, and to the development of a high-density SNP map.

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.

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 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.

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.

A contiguous clone map over 3 Mb on the long arm of chromosome 11 across a balanced translocation associated with schizophrenia.

Forty-nine clones derived by microdissection of a schizophrenia-associated t(1;11)(q42.1;q14.3) breakpoint region have been assigned by somatic cell hybrid mapping to seven discrete intervals on the long arm of human chromosome 11. Eleven of the clones were shown to map to a small region immediately distal to the translocation breakpoint on 11q. A 3-Mb contiguous clone map of this region was established by isolation of corresponding YAC recombinants. The contig was oriented and shown to traverse the translocation breakpoint by FISH and microsatellite marker analysis. This contig will facilitate the isolation of candidate sequences whose expression may be affected by the translocation.

Catch-linker + PCR labeling: a simple method to generate fluorescence in situ hybridization probes from yeast artificial chromosomes.

A simple and efficient method to generate hapten-labeled DNA fragments from a trace amount of YAC DNA isolated by PFGE is described. After agarase digestion of the gel slice containing the resolved YAC recombinant, the purified DNA is digested with Sau3Al and a compatible CL oligonucleotide duplex ligated on. A probe is generated by PCR amplification using a primer complementary to the CL with a single biotin moiety incorporated at the 5' end. When used as a FISH probe, this material yields mapping results superior to Alu-PCR or whole YAC labeling methods and allows sensitive detection of chimerism.

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.

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.

Role for the Wilms tumor gene in genital development?

Detailed molecular definition of the WAGR region at chromosome 11p13 has been achieved by chromosome breakpoint analysis and long-range restriction mapping. Here we describe the molecular detection of a cytogenetically invisible 1-megabase deletion in an individual with aniridia, cryptorchidism, and hypospadias but no Wilms tumor (WT). The region of overlap between this deletion and one associated with WT and similar genital anomalies but no aniridia covers a region of 350-400 kilobases, which is coincident with the extent of homozygous deletion detected in tumor tissue from a sporadic WT. A candidate WT gene located within this region has recently been isolated, suggesting nonpenetrance for tumor expression in the first individual. The inclusion within the overlap region of a gene for WT predisposition and a gene for the best-documented WT-associated genitourinary malformations leads us to suggest that both of these anomalies result from a loss-of-function mutation at the same locus. This in turn implies that the WT gene exerts pleiotropic effect on both kidney and genitourinary development, a possibility supported by the observed expression pattern of the WT candidate gene in developing kidney and gonads.