We wish to analyze the mechanisms responsible for the tissue-specific accumulation of alanine tRNAs in the silkworm, Bombyx mori. An understanding of the molecular basis of this phenomenon should provide insight into the mechanisms involved in eukaryotic cellular differentiation. Recent work in our laboratory has established that the constitutive and silkgland-specific types of alanine tRNA (which are nearly identical in sequence) are in fact encoded by distinct genes. This observation makes it unlikely that selective post-transcriptional modification accounts for the tissue-specific pattern of alanine tRNA production in vivo. In this proposal, we have focused on other levels at which tRNA synthesis might be regulated. Tissue-specific amplification or rearrangement of alanine tRNA genes, or, differential transcription of the two callses of tRNA genes are most likely to explain the selective appearance of the silkgland-specific type of alanine tRNA. The number and structure of alanine tRNA genes in genomic DNA from different silkworm tissues will be examined with hybridization probes that discriminate sharply between the two classes of genes. Tissue-specific changes associated with one or the other gene class should be easily detected by this method. To learn whether these genes are regulated at the level of transcription, we will compare the transcriptional properties of cloned representatives from each gene class in cell-free extracts from Bombyx ovaries and silkglands, as well as by microinjection of isolated genes into intact ovary and silkglands cells. By fractionating the Bombyx RNA polymerase III transcription apparatus, we will attempt to identify the component(s) responsible for any tissue-specific transcriptional differences we observe. We will also construct hybrid alanine tRNA genes in order to identify the nucleotide sequence responsible for functional differences between the constitutive and silkgland-specific type genes.
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