This is a continuing study on the molecular mechanisms of human Beta-IFN gene regulation. These studies involve the characterization of the properties of cis-acting DNA regulatory sequences and the identification and purification of trans-acting regulatory factors. The positive and negative regulatory DNA sequences will be precisely delineated by constructing and analyzing large numbers of single base mutations within the region already defined as being essential for Beta-IFN gene control. The properties of these regulatory sequences will be further investigated by studying their affects on heterologous promoters and enhancers. Interactions between cellular factors and specific Beta-IFN regulatory sequences will be studied using in vitro and in vivo DNAase footprinting methods. The in vitro footprinting method or other specific DNA binding assays will be used as a means of purifying the putative regulatory factors. Amino acid sequence information or antibodies raised against these purified proteins will be used to isolate the corresponding genes. These genes will then be characterized to derive the primary structure of the encoded proteins, and they will be used to produce large amounts of the proteins for use in DNA binding and in vitro transcription studies. Attempts will be made to isolate the genes encoding activating factors using differential hybridization methods, and the genes encoding Beta-IFN repressors will be isolated using a combination of genetic selection and gene rescue procedures. In order to study the mechanisms by which proteins can activate Beta-IFN gene expression, efforts will be made to establish transcription assays that are capable of supporting enhancer-dependent gene transcription. This problem will be approached by attempting to assemble transcriptionally active minichromosomes in Xenopus oocytes or in mammalian cells on extrachromosomal plasmids. The ultimate objective is to establish a fully reconstituted transcription system using purified cellular components that will mimic the in vivo regulation of the Beta-IFN gene.
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