A complementary DNA (cDNA) encoding for the mouse MyoD1 protein was isolated by Dr. A. Lasser and found to induce myogenesis in mesenchymal cells. Polyclonal antisera (rabbit) to bacterial fusion proteins containing the MyoD1 sequence were raised and showed that MYoD1 is a phosphoprotein present in the nuclei of proliferating myoblasts and differentiated myotubes, but not in nonmuscle cell types. The MyoD1 protein was found to contain a domain (22 amino acid) with marked similarity to a region in the myc-family proteins and to the predicted protein products of transcription units in Drosophila which are involved in neuronal determination and the development of nervous system. Based on the above information about the homologies of the MyoD1 protein shared by primitive myoblasts and neural transcription units, and on the knowledge that: (1) certain myogenous tissues (head and neck) originate embryologically from the neural crest, and (2) certain tumors of childhood contain both primitive neural and rhabdomyoblastic components (ectomesenchymoma, medullomyoblastoma), we will investigate the expression of the MYoD1 gene and protein in the RMS and PNET cell lines, to find out if the MyoD1 gene can be expressed in tissues not expressing a myogenous phenotype, but with the ability to do so. The study will employ Northern and Western blot analysis of the MYOD1 gene and protein correspondingly, and immunofluorescence using the polyclonal antisera against the MyoD1 protein to detect cytologic localization of the protein. The plasmids, carrying the MYOD1 gene and antisera will become available to us by Dr. A.Lasser who has already been contacted and agreed to provide us with the material. Moreover, the presence of the MyoD1 protein and the expression of the MyoD1 gene in these RMS cell lines before and after treatment with differentiating agents and TGF-b will be evaluated by immunofluorescence, Western blotting and Northern blotting correspondingly, to find out whether the MyoD1 gene and product can serve as a marker of differentiation in RMS.
