The goal of this project is to define the individual biochemical steps involved in the transcription and processing of eukaryotic messenger RNA. Experiments will include the following: 1. The development of a cell-free extract system capable of splicing mRNA is described. This system utilizes concentrated HeLa cell extracts, and a recombinant pBR322 derivative containing a segment of adenovirus-2 DNA which codes for an EIII region mRNA precursor containing two natural introns. The extract system is capable of synthesizing the mRNA precursor and removing the intron. Experimental procedures are described for: a. probing the exact sites at which the transcription of the precursor RNA begins and ends, and the sites of intron excision, b. improving the efficiency of transcription by modifying the plasmid by substitution of a strong promotor for the weak promotor, c. testing in vitro the splicing substrate quality of nuclear mRNA precursor made in vivo for the 30K adeno-SV40 hybrid protein and d. investigating the effects of inhibitors of transcription and processing. 2. Simultaneously, splicing of specific genes in vivo is being investigated using a eukaryotic viral cloning vehicle. Chimeric genes, having deletions, substitutions or rearrangements in regulatory elements in the EIII region, will be inserted into the virus. Such viruses will be used to infect human cells in culture, and the effects of the regulatory sequence changes on mRNA transcription and translation will be determined. Particular emphasis will be placed on the processing of mRNA at the 3' termini.
