A Glycine tRNA gene of rice embryos

Abstract

From an EcoRI library of rice DNA in pBR322, a clone bearing an insert of 7.6 kb hybridized with the ^32P-labelled tRNA prepared from germinating rice embryos. By restriction analysis and subsequent hybridization with the labelled probe, the tRNA gene was located within a 1.2 kb PstI fragment, which was sequenced by the dideoxy chain-termination method. A stretch of 71 nucleotides showed all the features of a eukaryotic tRNA gene, such as the cloverleaf structure with the appropriate invariant and semi-variant nucleotides and two internal control regions (ICRs). From the anticodon, it was identified as the tRNAGly_{Gly}Gly gene. This rice tRNA gene showed very high nucleotide sequence homology with nuclear-coded glycine tRNA genes from insect, rat, and human, and 100% homology with the tRNAGly_{Gly}Gly gene of wheat and Lupinus. By dot-blot analysis, the copy number of the rice glycine tRNA gene was determined to be about 5 per haploid genome. A 16 base pair (bp) sequence, TGTTTGTTTCAGCTTA, was repeated twice at 130 and 375 positions, and part of this sequence, TGTTTGTTT, was repeated three times at 130, 277, and 375. There were two T-stretches at +79 and +94, which may serve as transcription termination sites. The recombinant plasmid bearing the tRNAGly_{Gly}Gly gene was transcribed in a yeast cell extract into three transcripts of size 85, 80, and 74 nucleotides as determined on a sequencing gel. On prolonged incubation, the longer transcripts were converted into 74 nucleotide length, which corresponded to mature tRNA. Fingerprint analysis of the RNase T1_11 digest of ^32P UMP RNA transcripts showed 10 radioactive spots, which are expected from the nucleotide sequence of the tRNAGly_{Gly}Gly gene. This demonstrated that the tRNAGly_{Gly}Gly gene was faithfully transcribed in yeast cell extract. The deletion of the 5'-flanking region upstream to 544 did not have any effect on transcription. However, deletion of the 5'-flanking region from 262 onwards, including one 16 bp repeat sequence, showed a marked increase in transcription, implying a negative modulatory effect of the 5'-flanking region, probably due to the 16 bp repeat. Neither the 5'-ICR deletion mutant gene cloned in pBR322 nor the 3'-ICR deletion mutant gene cloned in M13mp19 could direct transcription, indicating that both of these elements are essential for transcription. However, the 5'-ICR deleted gene cloned in M13mp19 and M13mp18 could direct transcription, which may be due to homologous sequences present in the vector DNA. The homologous 5'-ICR-like sequences TGTTGTGTGG in M13mp18 and AAAACGACGG in M13mp19 vector DNAs are located at 73 and 26 bp, respectively, away from the 3'-ICR of the gene. The sizes of the transcripts from the M13mp18 clone were about 115 nucleotides, and from the M13mp19 clone were about 74, 80, and 85 nucleotides, showing the use of 5'-ICR-like sequences in the vector for transcription. In the 5'-ICR motif RGYNNARYGG (R = purine, Y = pyrimidine, N = any nucleotide), nucleotides R, G, Y, and A at positions 1, 2, 3, and 6, respectively, are highly conserved in all tRNA genes. However, the replacement of these nucleotides with A, A, and G at positions 2, 3, and 6 in the 5'-ICR-like sequence in the M13mp19 clone, and with T and T at positions 1 and 6 in M13mp18, did not affect transcription, implying that they may not be essential for promotion of transcription.