Patterns of intermediate filament (IF) protein expression have provided several model systems in which to study the regulation of both gene expression and morphogenesis during cell differentiation. During myogenesis in vitro, the expression of the vimentin gene decreases, whereas expression of desmin is induced upon myoblast fusion and partially replaces vimentin expression during terminal differentiation. We have previously shown that the developmental regulation of these IF subunits is controlled at the mRNA level. C2 myoblasts transferred with the oncogenic ras genes lose their ability to differentiate. We have recently found that these transformed cells, when induced, fail to appropriately down regulate vimentin and up regulate desmin mRNAs. How are these two phenomena related? At what level is the normal and disrupted developmental expression of vimentin regulated? Is it at the transcriptional or post-transcriptional level? What is the role of these two proteins in this differentiating system and how does it correlate with their expression pattern? This proposal will examine some of the above issues. We will determine the level of regulation of vimentin and desmin expression during normal and disrupted mouse myogenesis by measuring initial transcription rates and mRNA stabilization. Using recently developed gene transfer techniques, we will investigate possible modes of function of these two proteins during muscle differentiation. The following lines of investigation will be employed. cDNA or genomic constructs designed to either express high levels of vimentin or desmin at the wrong stage during myogenesis or produce high levels of antisense RNAs (which will arrest the expression of the endogenous gene), will be introduced into myogenic cells. This will allow us to study: A) any possible dependence of the expression of these to genes upon each other; and b) the functional consequences of such forced or inhibited expression on muscle differentiation. For these antisense experiments, we need the corresponding mouse DNAs which will be isolated by using the available chicken probe. Alternatively, we will use antisense oligonucleotides to inhibit the translation of these proteins. Eventually, antisense constructs as well as constructs that will overproduce vimentin or desmin will be used in mouse transformations and the consequences of the absence or over accumulation of these proteins in normal and abnormal muscle function will be investigated in vivo.
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