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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032851-03
Application #
3282016
Study Section
Molecular Biology Study Section (MBY)
Project Start
1983-12-01
Project End
1986-11-30
Budget Start
1985-12-01
Budget End
1986-11-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Oregon
Department
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Martinez, M Juanita; Sprague, Karen U (2003) Cloning of a putative Bombyx mori TFIIB-related factor (BRF). Arch Insect Biochem Physiol 54:55-67
Ouyang, C; Martinez, M J; Young, L S et al. (2000) TATA-Binding protein-TATA interaction is a key determinant of differential transcription of silkworm constitutive and silk gland-specific tRNA(Ala) genes. Mol Cell Biol 20:1329-43
Ouyang, C; Sprague, K U (1998) Cloning and characterization of the TATA-binding protein of the silkworm Bombyx mori. Gene 221:207-13
Young, L S; Ahnert, N; Sprague, K U (1996) Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage. Mol Cell Biol 16:1256-66
Smith, T P; Young, L S; Bender, L B et al. (1995) Silkworm TFIIIA requires additional class III factors for commitment to transcription complex assembly on a 5S RNA gene. Nucleic Acids Res 23:1244-51
Dunstan, H M; Young, L S; Sprague, K U (1994) TFIIIR is an isoleucine tRNA. Mol Cell Biol 14:3588-95
Dunstan, H M; Young, L S; Sprague, K U (1994) tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage. Mol Cell Biol 14:3596-603
Sullivan, H S; Young, L S; White, C N et al. (1994) Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions. Mol Cell Biol 14:1806-14
Young, L S; Dunstan, H M; Witte, P R et al. (1991) A class III transcription factor composed of RNA. Science 252:542-6
Young, L S; Rivier, D H; Sprague, K U (1991) Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors. Mol Cell Biol 11:1382-92

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