The project described in this application employs mammalian somatic cell and molecular genetics to recognize and characterize a novel set of human structural gene and regulatory mutations. As for all mutant surveys, the ultimate value of this project depends directly on the nature of mutants isolated. Because many factors that usually constrain the spectrum of viable mutants observed are obviated by genetic design of this experimental system, and because a comprehensive set of available nucleic acid reagents facilitate the required molecular analyses, mutants isolated are likely to comprise a novel and informative genetic resource. Application of current molecular genetic technology to several important medical (as well as agricultural and industrial) problems awaits significantly better understanding of mechanisms regulating mammalian gene expression. Information expected from this project is likely to contribute significantly to the required body of knowledge. A set of """"""""wildtype"""""""" cell lines that contain a coresident pair of functional, ribosomal protein (RPS14) alleles has been constructed using DNA transfection. The genes used are unique in that their expression results in selectible drug resistance phenotypes and their complete nucleic acid sequences are known. RNA and protein products of the two alleles can be resolved by convenient molecular assays. Thus, mutations which alter either gene's product (structural gene mutations) can be distinguished from mutations that disrupt their normal patterns of expression (regulatory mutations). Because the """"""""wildtype"""""""" cells maintain two functionally regulated copies of the gene, they can express mutations which in normal cells would be lethal. One copy of the gene can be rescued from mutant clones; and these can be mapped to specific nucleic acid sequences. Substantial biochemical and genetic data from lower eu- and prokaryotic systems indicate that ribosomal protein genes are stringently regulated by the combined action of transcriptional and post-transcriptional mechanisms. Experiments to distinguish which of these operate on a human ribosomal protein gene and to discern cis-active regulatory sites within the human gene are described.
