The CTLA4 gene is important for T lymphocyte-mediated immunoregulation and has been associated with several autoimmune diseases, in particular, type 1 diabetes. To model the impact of natural genetic variants of CTLA4, we constructed RNA interference (RNAi) ''knockdown'' mice through lentiviral transgenesis. Variegation of expression was observed in founders but proved surmountable because it reflected parental imprinting, with derepression by transmission from male lentigenics. Unlike the indiscriminate multiorgan autoimmune phenotype of the corresponding knockout mice, Ctla4 knockdown animals had a disease primarily focused on the pancreas, with rapid progression to diabetes. As with the human disease, the knockdown phenotype was tempered by genetic-modifier loci. RNAi should be more pertinent than gene ablation in modeling disease pathogenesis linked to a gene-dosage variation.autoimmune diabetes ͉ imprinting ͉ lentiviral transgene ͉ RNAi ͉ variegation

Bacteriophage PM2 DNA is a 10‐kb covalently closed circular (ccc) molecule with a reported superhelical density of sigma = ‐0.12. Here we describe the binding of anti‐Z‐DNA antibodies to PM2 form I DNA under high and low salt conditions. The binding to PM2 DNA has been demonstrated by competitive radioimmunoassay (RIA), retardation of the DNA:antibody complexes in agarose gels and visualization by electron microscopy. The antibody binding is dependent on the degree of negative supercoiling. Thus, PM2 form II and form III did not bind the antibody. The low salt RIA results indicated the presence of 200‐400 bp of left‐handed DNA per PM2 molecule. This could reduce the effective superhelical density to sigma = ‐0.04 to ‐0.08, a range comparable with those found for other ccc DNAs in vivo. Electron microscopy revealed that a maximum of 22 antibody molecules bind to PM2. Single‐site restriction with HpaII of the fixed DNA:antibody complex showed a cluster of four to five antibody molecules bound near one end of the linear DNA molecule. The evidence presented indicates that PM2 DNA contains regions of left‐handed conformation under physiological conditions (low salt concentration) as well as at high salt concentrations. In addition, electrophoretic analyses of PM2 topoisomers indicate the presence of left‐handed regions at superhelical densities less than that of isolated PM2 DNA.

Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy. The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule. A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05-0.18 of the single Hpa II restriction site. Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content. A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites.