Unregulated levels of myc protein, the gene product from the c- myc proto-oncogene, are causal in the progressive development of malignant transformation in a variety of animal and human tumors. The focus of this project is to study the regulation of two processes which are important for determining the amount of myc protein in cells: c-myc transcription and c-myc mRNA stability. The proposed study will elucidate normal regulatory events and will also reveal how disruption of particular mechanisms can result in malignant transformation. The overall approach is to dissect biochemically and genetically the function of individual proteins with the ultimate goal of reproducing regulated c-myc transcription and mRNA degradation from purified components in vitro. We also wish to elucidate the molecular nature of the nucleic acid-protein and protein-protein interactions involved. Transcriptional regulation involves multiple proteins, most currently unknown, which somhow interact to form stable initiation complexes. Two c-myc transcription factors, previously identified in this lab, will be purified so that cDNAs encoding them can be cloned and expressed. Availability of cDNA clones and purified protein will allow the transcriptional activity of these factors to be studied in vitro and the nature of their interactions with DNA and other proteins to be determined. We will also study their regulated expression using both cDNAs and antibodies. In addition, all proteins binding 5' of the c-myc transcription start sites and in the transcription pausing region will be identified and subsequently analyzed for differential activity when c-myc transcription is modulated. Additional factors will be studied, as described above. Very little is understood regarding mRNA stability. Recently, this lab has developed a simple in vitro system which reproduces the regulated degradation of c-myc mRNA observed in vivo. This puts us in a strong position to identify, purify and characterize factors which are responsible for regulated c-myc mRNA degradation.
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