Myogenesis provides a model system to examine the control of contractile protein biosynthesis. Alpha Actin is found only in skeletal muscle, while other polymorphic forms of actin are found in a large variety of nonmuscle cell types. Nonmuscle actins are associated with cell cytoskeleton, movement, division, adhesion, shape and excretion. In the case of muscle Alpha actin, this p rotein appears only after myoblast fusion and the controlling factor is unknown. The major objective is to examine the mechanisms(s) involved with the selective expression of the skeletal muscle Alpha actin structural gene from all other members within the actin multigene family, during muscle development in culture. Two dependent lines of investigation will be employed. First, the actin structural genes within the chicken genome will be isolated, identified, and then characterized for homologous and nonhomologous nucleic acid sequences in comparison to the muscle Alpha actin gene. It will be necessary to subclone genomic actin sequences into plasmid vectors and then restriction endonuclease map the actin genes. The conserved coding region, transcriptional oreintation and location of intervening sequence in the actin genes will be determined. The Alpha actin structural gene will be nucleic acid sequenced as well as portions of the other actin genes. Second, specific actin gene sequences will be used as hybridization probes to investigate transcriptional regulation of the Alpha actin structural gene product during myogenesis. Initial transcription rates and stability of Alpha actin mRNA will be determined by pulse chase analysis. The role of DNA modification and conformation will be examined as correlates to gene activity. Finally, the long range goal will be to identify potential regulatory sequences in the Alpha actin gene which might allow for its selective expression in muscle. In this way, it might be possible to understand the defects in gene regulation in inherited neuromuscular diseases.
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