Sequence Organization and Expression of Mouse Trna Genes

Harding, John

Abstract

We propose to examine the sequence organization and regulation of specific families of mouse tRNA genes. These goals will be accomplished by: 1) Sequencing the majority of mouse genes coding for tRNAHis, previously isolated in recombinant lambda phages in our laboratory. 2) Characterizing the tRNAHis gene transcripts synthesized in mouse cells in vivo. 3) Constructing deletion and point mutants of the flanking regions of various mouse tRNA genes and analyzing the effects of the mutations on transcription in vitro. The mutated genes will include a) tRNAHis genes, one of which has been demonstrated by us to contain a 5' flanking sequence inhibitory to transcription. b) tRNAAsp genes which contain homologous 5' flanking regions. c) a tRNAiMet gene which has a 5' flanking region homologous to that of a human gene. 4) Cloning selected tRNA gene mutants into vectors which can be introduced into mammalian cells and analyzing the in vivo expression of the genes. These experiments will provide novel information about the following topics: 1) The sequence heterogeneity of a dispersed reiterated eucaryotic gene family. 2) The relationship of sequence heterogeneity to gene expression. 3) The specific DNA loci involved in control of expression of eucaryotic genes in vitro and in vivo. 4) The significance of 5' flanking region homologies which, in some cases, have apparently been conserved since the mammalian rediation.