Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder affecting 1 in 3500 males. A less severe and less common form is Becker muscular dystrophy (BMD). Only recently has the basic defect in DMD and BMD been recognized: a region on the human X chromosome is disrupted by mutation. The primary transcript of the region was detected, and cDNA clones were isolated that encompassed the entire transcript. The sequence of the encoded protein was predicted from cDNA nucleotide sequence, portions of the protein were overexpressed in bacterial cells, and these peptides were used to generate immunoreagents against the DMD gene protein, dystrophin. The molecular genetic identification of this protein via analysis of mutations found in patients' material has led to a means of improved diagnosis of DMD/BMD in affected individuals and their family members. The severely affected DMD patients have little or no detectable dystrophin in their muscle, whereas BMD patients have nearly normal concentrations of an altered form of dystrophin; patients with all other neuromuscular diseases have normal dystrophin.

Part of the higher-order structure of chromatin is achieved by constraining DNA in loops ranging in size from 30 to 100 kilobase pairs; these loops have been implicated in defining functional domains and replicons and possibly in facilitating transcription. Because the human active and inactive X chromosomes differ in transcriptional activity and replication, we looked for differences in their chromatin loop structures. Since the islands of CpG-rich DNA at the 5' ends of X-linked housekeeping genes are the regions where functional differences in DNA methylation and nuclease sensitivity are found, we looked for scaffold association of these sequences after extraction of histones with lithium diiodosalicylate. Specifically, we examined the 5' CpG islands within the hypoxanthine phosphoribosyltransferase, glucose 6-phosphate dehydrogenase, P3, GdX, phosphoglycerate kinase type 1, and alpha-galactosidase loci in human lymphoblasts obtained from individuals with 1 to 4 X chromosomes. Although we detected no scaffold-associated regions near these genes, we found several such regions at the ornithine transcarbamylase and blood clotting factor IX loci. Our results suggest that the CpG islands are excluded from the nuclear scaffold and that even though transcriptionally active, housekeeping genes are less likely than X-linked tissue-specific genes to be scaffold associated. In all cases, the pattern of scaffold association was the same for loci on active and inactive X chromosomes.

JC virus (JCV) is a ubiquitous human papovavirus that shares sequence and structural homology with simian virus 40 (SV40). In contrast to SV40, expression of JCV is restricted to a small number of cell types, including human fetal glial cells, uroepithelial cells, amnion cells, and some endothelial cells. To study the control of JCV early region expression, we made heterokaryons and stable hybrids between JCV-transformed hamster glial cells and mouse fibroblasts. Binucleate heterokaryons exhibited extinction of large tumor antigen expression in the hamster nuclei as assayed by indirect immunofluorescence. This extinction was both time and dose dependent: extinction reached maximal levels at 24-36 hr after fusion and was dependent on the ratio of glial cell to fibroblast nuclei in multinucleated heterokaryons. Extinction also was observed in stable hybrids between the glial cells and mouse Ltk- cells. Southern blot analysis showed that the extinguished hybrids contained viral sequences. Reexpression of large tumor antigen was observed in several subclones, suggesting that extinction was correlated with the loss of murine fibroblast chromosomes from these hybrids. The cis-acting region that mediates extinction resides within the viral regulatory region, which contains two 98-base-pair repeats that have enhancer activity. These data demonstrate that cellular factors that negatively regulate viral gene expression contribute to the restricted cell-type specificity of this virus.

In mammals, X-chromosome dosage compensation is achieved by inactivating one X chromosome in female cells. To test the hypothesis that genes on the silent X chromosome reactivate as a consequence of ageing, we examined the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 41 women who are heterozygous for mutations at this locus, leading to severe deficiency of the enzyme (Lesch-Nyhan syndrome). We find that heterozygotes who are more than 10 yr old have an excess of HPRT+ skin fibroblast clones (59% rather than the 50% expected as a consequence of random X inactivation) but this excess does not increase with age. Further studies of eight of these heterozygotes show that the silent locus does not detectably reactivate spontaneously in culture, but only in response to treatment with 5-aza-2-deoxycytidine, a potent inhibitor of methylation. There is no age difference in the frequency of this reactivation as assayed by HATr clones, and a more sensitive autoradiographic assay shows only a twofold difference between young and old heterozygotes. Thus, age-related reactivation is not a feature of all X-linked loci, and may have species, tissue and locus-specific determinants.

To determine if expression of genes on the inactive X is inducible in human cells, we looked for reactivation events in a clone of fibroblasts transformed with origin-defective SV40. The karyotype of these cells was grossly heteroploid so that the aneuploidy associated with SV40 transformation occurs even in the absence of viral replication. This transformed clone, heterozygous for hypoxanthine phosphoribosyltransferase (HPRT), lacks HPRT activity, as the mutant allele is on the active X and the normal allele on the inactive X. Reactivation of the HPRT+ allele on the inactive X was observed at a frequency of 6 X 10(-5) per cell and increased approximately eightfold following treatment with the cytidine analogs 5-azacytidine (5azaC) and 5-azadeoxycytidine. The fact that spontaneous reactivation is detectable in some clones, but not all, suggests that the environment of the SV40-transformed cell, although not sufficient to induce generalized derepression, increases the frequency of rare reactivation events. The methylation pattern at the HPRT locus revealed transformation-associated alterations that may have predisposed these cells to reactivation events, spontaneous as well as 5azaC-induced.

We have described the development of a selective system useful for the identification and characterization of onc genes capable of conferring growth factor independence. The use of defined, serum-free media allows us to select for transformed cells which have lost specific growth factor requirements. We have used this system to show that several cloned onc genes generate different transformed phenotypes with respect to growth factor requirements. BPV-1 is active in relieving contact inhibition, yet these transformed NIH/3T3 cells retain their stringent requirement for FGF. In contrast, sis and H-ras were equally proficient at relieving contact inhibition and the requirement for FGF. Sis induced equal numbers of colonies regardless of the presence or absence of insulin, however, H-ras-mediated colony formation decreased four-fold when insulin was removed. This suggests that H-ras is less efficient in relieving the insulin requirement than is sis. To determine if colony formation by H-ras is a function of dosage, we are conducting experiments to measure the level of expression of p21 in transformants selected with and without insulin in the media. We have also presented data to show that loss of contact inhibition and loss of growth factor requirements are dissociable phenotypes under separate control in some cells. Thus, it should be possible to use this selective system to identify transforming genes in tumor DNA. Since some of these genes may be undetectable by the standard focus forming assay, selection in MSF medium may prove to be a useful tool for identifying and elucidating the action of activated cellular onc genes.