P.F. Jacobsen

VARIATION IN THE METHYLATION PROFILE AND STRUCTURE OF PAX3 AND PAX7 AMONG DIFFERENT MOUSE STRAINS AND DURING EXPRESSION

Structural alterations within the myogenic and neurogenic developmental gene Pax7 which involve TaqI recognition sequences have previously been reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pax genes which may influence skeletal muscle regrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse strains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the paired box. Differences in the size of MspI and HpaII fragments hybridising with Pax7 and Pax3 region specific sub-probes indicate that the paired boxes are hypomethylated, whereas the region encoding the homeodomain of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, which expresses Pax7 but not Pax3, the homeodomain encoding region of Pax7 is hypomethylated. In spleen cells, the Pax7 paired box is transcribed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pax genes may be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for CpNpG methylation within and immediately downstream from the region encoding the homeodomain. Interestingly, CpNpG methylation remains when the Pax7 homeobox is expressed. Structural variation recognised by MspI digestion and differences in the methylation profile of Pax7 are not associated with the ability to regrow damaged skeletal muscle.

Mesenchymal Differentiation of Cell Lines Obtained From Human Gliomas Inoculated Into Nude Mice

Tumor formation in nude mice (nu/nu Balb c outbred) inoculated with cells from four new permanent human glioma cell lines was studied. Three of these lines had previously been shown to display features of striated muscle in vitro. Histochemical and immunochemical techniques together with electron microscopic study confirmed that striated muscle differentiation continued to be expressed in vivo. Two of the cell lines arguably showed greater striated muscle differentiation in vivo, whereas one has lost this ability. In one of the two, further mesodermal differentiation was evident with the formation of cartilage.

Genetic polymorphism of the murine myogenic gene Myo-D1

Polymorphism of the myogenic gene, Myo-D1, has been sought to examine genetic mechanisms which control skeletal muscle development. By Southern analysis, three restriction-fragment length polymorphisms (RFLPs) have been found in various mouse strains using the TaqI, SacI and BglII restriction endonucleases and a full-length cDNA Myo-D1 probe. Reference to the distribution of RFLPs in different mouse strains derived from Mus mus (M.m.) domesticus and M.m. musculus subspecies suggests that Myo-D1 rearrangements are subject to nonrandom association. The biological significance of RFLP of the Myo-D1 gene is yet to be determined.