The nature and position of transcriptional control elements responsible for the differential control of gene expression in eukaryotic cells have not been precisely defined. A primary goal of this section is to develop new methods to investigate transcriptional control mechanisms, which are mediated by nucleic acid promoter elements, utilizing an active in vitro transcription system. Tissue-specific promoter elements will be used to stimulate gene expression in vivo utilizing retroviruses as mediators of specific gene transfer. To identify components required for transcriptional of mRNA by RNA polymerase II, distinct transcriptional complexes have been characterized using newly developed techniques of DNA competition and have been purified using newly developed techniques of DNA affinity chromatography. Proteins associated with specific DNA sequences following affinity purification are being used to identify: 1) Transcription factors and their functionality and 2) DNA control sequences and topology necessary for regulated initiation and elongation of in vitro transcriptional intermediates. Specific synthetic control sequences of DNA are being assembled, based on the presence of tissue-specific transcription factors and nucleic acid topology. These synthetic promoters are being used to stimulate expression and secretion of the genes for adenosine deaminase, rat growth hormone and ECGF in endothelial, and liver cells, respectively, following cloning into retroviruses. Both in vitro and in vivo infections with these modified expression vectors are being studied as models of gene transfer into genetically deficient cells.
