This research deals with production, and subsequent destruction of PDGF- inducible mRNAs. We have two broad objectives. Our first objective is to identify nuclear and cytoplasmic components of a PDGF signal transduction pathway which does not involve protein kinase C or the cis acting """"""""Serum- Response Element"""""""". Towards this end, we will carry on with structure/function analysis of the promoter for """"""""JE""""""""- a gene which is induced by PDGF through activation of the kinase C-independent pathway. Transient transfections have shed little insight into functional elements of the JE promoter. Accordingly, we will develop new tactics. We will ligate the JE promoter to a reporter gene which can be detected by fluorescence activated cell sorting (FACS). Through FACS, we hope to conduct structure/function analysis of the JE promoter in stable transfectants without resorting to dominant selectable marker genes. At the cytoplasmic level, we will evaluate the role of c-raf protein as an intermediate in JE induction. These experiments will involve cell microinjection and in situ hybridization. The second broad objective is to define cis and trans acting elements which mediate selective degradation of PDGF-inducible mRNAs. Here we will also develop new technologies. Artificial mRNAs will be microinjected into the cytoplasm of Balb/c-3T3 cells to define cis acting elements which regulate mRNA stability. We have adapted """"""""RNA gel-band shifts"""""""" to the problem or trans acting factors involved in message stability. Using RNA band shift, we are isolating and characterizing a protein which binds specifically to 3' A/U rich motifs within PDGF-inducible mRNAs. We will explore the potential of yeast genetics as an alternative vehicle to the isolation of trans acting factors.
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