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Studies on the valine transfer RNAs and their genes in Drosophila melanogaster

University of British Columbia

The coding properties of the 3 major valine tRNA isoacceptors of Drosophila melanogaster, the nucleotide sequences of tRNA[sub=Val, sub=3b] and tRNA[sub=Val, sub=4] and the nucleotide sequences of genes for these two tRNAs have been determined. Valyl-tRNA[sub=Val, sub=3a] binds strongly to ribosomes in response to the trinucleotide GUA and to a lesser extent with GUU and GUG. Valyl- tRNA[sub=Val, sub=3b] binds strongly in the presence of GUG and very weakly with the other 3 triplets whereas valyl- tRNA[sub=Val, sub=4] binds strongly in the presence of GUU, GUC, and GUA and weakly with GUG. The nucleotide sequences of tRNA[sub=Val, sub=3b] and tRNA[sub=Val, sub=4] were determined by a combination of techniques. For both tRNAs most of the sequence was determined by the method of Stanley and Vassilenko. The sequences at the 5' and 3'-ends of the molecules were determined by wandering-spot analysis. Regions of the molecules that could not be sequenced by these two techniques were determined by the gel read-off method. The use of tRNA modified with chloroacetaldehyde to overcome problems in sequencing RNA by the gel read-off method caused by secondary structure in the RNA is described. The nucleotide sequence of tRNA[sub=Val, sub=4] is: GUUU[sub=m]⁷CCGUm¹GGUG ѱAGCGGDU (acp³ U)AUCACA1ѱCUGCC[sub=m]UIACAm⁵CGCAGAAGm⁷GCCCCCGGѱC Gm¹ AUCCCGGGCGGAAACACCA. About 50% of the U residues at position 20 are modified to acp³U. One of the C residues at position 48 or 49 is probably modified to m5C. The nucleotide sequence of tRNA[sub=Val, sub=3b] is: GUUUCCGѱAGUGS1 AGCGGDacp³ UAUCACGѱGUGCUUC ACACGCACAAGm⁷- GDCCCCGGTѱCGm¹ AACCC GGGCGGGAACACCA. The C residue at position 48 is probably modified to m⁵C. The observed codon responses of the two tRNAs are discussed in relation to the anticodons found. Val The two tRNA[sub=Val, sub=4] genes of the recombinant plasmid pDt55 were sequenced by the Maxam and Gilbert method. This plasmid hybridizes to the 70BC site on the polytene chromosomes, a major site of tRNA[sub=Val, sub=4] hybridization. The two genes are of opposite polarity and are separated by 525 bp of DNA. The genes have identical sequences, which correspond to that expected from the sequence of tRNA[sub=Val, sub=4]. The nucleotide sequence of the tRNA[sub=Val, sub=3b] gene of recombinant plasmid pDt78R was also determined. This plasmid hybridizes to the 84D site, a major site of tRNA[sub=Val, sub=3b] hybridization. The sequence of the gene corresponds to that expected from the sequence of tRNA[sub=Val, sub=3b]. Comparison of the valine tRNA genes sequenced in this study and those determined by other workers shows that tRNA genes from major sites of tRNA[sub=Val, sub=3b] or tRNA[sub=Val, sub=4] hybridization to polytene chromosomes correspond exactly to the tRNA[sub=Val] sequences while tRNA tRNA[sub=Val] genes from minor sites of tRNA hybridization differ at 4 positions from the sequences expected on the basis of the tRNA sequences. The possible significance of this finding is discussed.

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2010-04-01

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http://hdl.handle.net/2429/23294

Biochemistry and Molecular Biology

University of British Columbia

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