The purpose of this project is to study human gene organization, structure and expression by detailed analyses of selected human tRNA genes. Since the structures of mammalian tRNA genes and their arrangement in mammalian genomes have not been elucidated, the purpose of this research is to thoroughly characterize the structures (nucleotide sequences) of several human tRNA genes (as well as their corresponding mammalian tRNAs) to elucidate their unique features, since the mode of organization and expression of tRNA genes in mammals (especially humans) may differ greatly from those tRNA genes from lower eukaryotes which have been studied (such as Drosophilia and yeast). Representative tRNA genes will be isolated by recombinant DNA techniques, using radioactively labeled mammalian tRNAs to screen human gene libraries harbored in lambda bacteriophages, and their nucleotide sequences determined. The nature and mode of gene expression of the selected human tRNA genes will be investigated by studying the precise precursor/product relationships of the nascent RNA transcripts of these genes by RNA sequence analyses of in vitro transcription products derived from a homologous HeLa cell-free system.
