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.

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.

CpG islands surround a DNA segment located between translocation breakpoints associated with genitourinary dysplasia and aniridia.

We have isolated a DNA segment absent from all the constitutionally deleted chromosomes 11 of our patients with Wilms tumor. This marker separates two balanced translocations that break in band 11p13: the distal one associated with aniridia (AN2), and the proximal one with genitourinary dysplasia (GUD). The GUD breakpoint maps within the smallest region of overlap (SRO) for the Wilms tumor (WT) gene locus, thus strengthening the previous suggestion of an association between Wilms tumor and other abnormalities of the genitourinary system. The 11p13 translocation breakpoint associated with T-cell acute lymphatic leukemia (T-ALL) is centromeric to the SRO and separated from the WT locus by at least one known gene. This region of the human genome (11p13) is rich in CpG islands that potentially identify genes, some of which may be involved in the various phenotypes associated with the WAGR syndrome. This is consistent with the proposition that the majority of human genes are in G-negative bands.

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.