Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan 11529, ROC
5s rRNA is one component of the large subunit of ribosomes. Genes coding for 5s rRNA are located on chromosomes 7, 9 and 11 (Chung et al. 1993). The interspacious region (spacer) of the 45s rRNA genes have substantial variation among wild rice species. In this study, we tried to detect any variation that might exist in the spacer of 5s rRNA genes in the seven rice genomes, namely AA, BB, CC, BBCC, CCDD, EE and FF.
Hariharan et al. (1987) reported that 5s rRNA genes are present in tandem repeats of 0.3 kb sequence in the genome of cultivated rice. There is however no homology between the 184 bp spacers of the 5s rRNA genes in rice and those is other grain crops.
For analysis of the 5s rRNA spacer, polymerase chain reaction (PCR) amplification was conducted in the GeneAmp PCR System 9600, using genomic DNAs from the various genomes as template and the two end sequences of the repeat unit of the known 5s rRNA as primers. The PCR products were ligated into pGEM-T vector and transformed into competent cells of E. coli JM109 using the E. coli Pulser. Transformed bacteria were inoculateld onto plates of LB medium with ampicillin, Xga1 and IPTG. Only white colonies were picked and inoculated into 5 ml culture of LB liquid medium with ampicillin for plasmid DNA isolation. Analysis of the insert size of the recombinant clones was carried out by Southern blot analysis after digestion of the plasmid DNA with ApaI and SacI next to the ligation sites T on the PGEM-T vector.
Southern blot analysis of the PCR products amplified from the seven genomes indicated that the major bands varying from 0.28 kb to 0.52 kb in size correspond to the unit repeats of the 5s rRNA genes. One unit sequence of all genes consists of 119 bases, starting with GGA and ending with CCC except for the clone W0045-23 (genome BBCC) which is GGT and CCT, respecively. Length heterogeneity was therefore detected for the spacers of the 5s rRNA genes amplified from the various genomes. These were 20, 24 and 146 bases in excess, respectively, in the spacer of the clones W0107-4 (genome AA), W1057-9 (FF) and W1263-1 (CC) compared with that of IR20 (AA) which was used as the control in the experiments and had 184 bases. On the other hand, three clones representing genomes BBCC, EE and CCDD had fewer bases, ranging from 161 to 176 bp. Base identity was high (86%) in genomes AA, and FF but low (40%) in BBCC, EE and CCDD.
Within the extent of spacer, there was a rRNA-like segment of 91 bases. Length heterogeneity was also detected in this segment. An addition of 26 to 43 bases to the tRNA-like segment of clones W0107-4(genome AA), W1057-9(FF), W0008-3(EE) and W1263-1(CC) but a substraction of 30 bases from clone W0045-23(BBCC) were found. In the case of clone W0613-6(CCDD), an addition of 15 bases and a substraction of 20 bases were involved. Thus, the overall length of W0613-6 tRNA-like segment was 5 bases shorter than the control that had 91 bases. Base identity was high (87%) in clones W0107-4(AA) and W1057-9(FF) but low (30 to 50%) in the remaining representative clones. As a result, the secondary structure of the tRNA differs by forming a new stem of 26 bases in the clover-leaf in the genomes AA, CC, EE, and FF or forming a clover-leaf having only three loops, instead of five in the genome BBCC.
Within the spacer, a putative TATA box (ATATA) can be found in genome AA and FF, but a TATA-like box (ACAAATG, identical to that in Brassica campestris) in genome EE, and a TATA-like box (ACGCTA) in genomes BBCC and CCDD, and a TATA-like box (TGCGCGT) in genome CC were found instead. The transcription termination signal, a cluster of five or six T flanked by a G at both sides is conserved in all the genomes. In addition, there are two T clusters with C and one cluster of four T without C.
In the middle of the 119 bp coding region of the 5s rRNA genes, the well-known GC motif, with a conserved pentanucleotide core sequence and a larger consensus sequence, TGGGCGAGAGT which is known to be involved in regulation of transcription in humans and in Brassica campestris, is present in all the genomes examined.
The sequence of at least one more positive band amplified from each genome and probed by the 5s rRNA gene remains to be analysed.
References
Chung, M. C., C. N. Ning and H. K. Wu, 1993. Localization of ribosomal RNA genes on rice chromosomes. Bot. Bull. Acad. Sin. 34: 43-55.
Hariharan, N., P. S. Reddy and J. D. Padayatty, 1987. 5s rRNA genes in rice embryos. Plant Mol. Biol. 9: 443-451.