To analyze the molecular mechanisms involved in selective gene expression in differentiated eukaryotic cells, we are studying the alanine tRNA genes from the silkworm, Bombyx mori. These genes are interesting because their products, alanine tRNAs, are accumulated in a tissue-specific fashion. One species of alanine tRNA (tRNACAla) is present in all tissues and is therefore designated constitutive, while the other species (tRNASGAla) is found only in the silkgland. Our goal is to discover the molecular basis for the tissue-specific appearance of alanine tRNA in the silkworm. An investigation into the mechanism responsible for this phenomenon addresses the general problem of gene control during eukaryotic cellular differentiation. We think it likely that tissue-specific regulation of polymerase III transcription is the basis of the differential accumulation of the two alanine tRNAs. We have discovered both cis-acting and trans-acting elements that differentially affect transcription of these templates. Because these effects are large, and because they can be analyzed in vitro, we are in a strong position to determine the precise molecular basis of differential tRNAAla gene expression. We propose a detailed in vitro analysis of the components that interact in trans with the tRNAASG1a gene. In brief, we will define the transcription component(s) that differentially affects transcription of tRNACAla and tRNASGAla genes. We will ask whether the gene-specific component exerts its effect by binding to the gene itself, or by binding to part of the general transcription apparatus, and we have proposed experiments to determine what step in the overall transcription reaction is differentially affected on the two templates. To determine the relevance of our in vitro analysis to the situation in vivo, we will use in vivo footprinting to examine transcription complexes formed in intact silkworm cells. In addition, with antibodies raised against gene-specific transcription components, we will determine the tissue distribution of these factors to see if modulations in their level could account for the activity of tRNASGAla genes in the silkgland.
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