Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, U.S.A.
In rice, it has been estimated that approximately 50% of the genome is comprised of repetitive sequences (Deshphane and Ranjekar 1980). Several of these sequences have been cloned (Wu and Wu 1987; Zhao et al. 1989), and their genome locations have been characterized by in situ hybridization and PFGE (Wu et al. 1991; Wu et al. 1994). Among them, Os48, OsG430, and Osc567 were found mostly at euchromatic ends of either several or all 12 five chromosomes and were physically linked to the telomeres. Another repetitive element OsG756, however, was located at heterochromatic short arms and at regions flanking all the centromeres by in situ hybridization (Wu et al. 1991). In this paper, we report the isolation of a new rice repetitive sequence, OsG340, which has a repeat unit of 340 bp. By using PFGE combined with rare-cutting restriction enzymes, we have studied the genome organization of OsG340 and OsG756.
OsG340 was isolated accidentally in the process of characterizing tRNA genes in rice. A rice tRNAgly gene and 400 bp of its 5'-region were isolated from total DNA by polymerase chain reaction (PCR) based on the published sequence (Reddy and Padayatty 1988). This 500-bp PCR fragment was cloned in pbluescript vector (Strategene) at an EcoRV site and was used as the probe to screen a rice (cv. IR36) genomic library. Eighty positive clones were plaque-purified, and DNA was prepared from these clones and analyzed by digestion with EcoRI restriction enzyme. Southern blot hybridization with the tRNAgly gene probe identified five hybridizing fragments of 5-6 kb in size. Further restriction mapping, with various restriction enzymes followed by subcloning in pbluescript, yielded four clones containing the OsG340 repetitive sequence. These clones, designated as OsG340-1, -12, -21, and -31, had rice DNA inserts of 0.5 kb, 1.2 kb, 1.3 kb, and 1.8 kb, respectively. The inserts were sequenced by the dideoxynucleotide-chain-termination method, and the portions of the OsG340 repeats were
Fig. 1. Comparison of OsG340 sequences from rice. Complete sequence is shown
for four OsG340 elements isolated from the rice (cv. IR36) genomic library and
the consesus sequence (OsG340-0). These elements are shown with their phage
clone numbers. Gaps are introduced, when necessary, in order to maintain
alignment of homologous sequences. The sequence for pOsG340 has been
deposited in the Genome Sequence Data Base, National Center for Genome
Resources, under the accession number L36562.
compiled and aligned by the GCG DNA sequence analysis program (Devereux et al. 1984).
Fig. 1 displays the alignment of the sequences. The regions of OsG340 repeats were compared with each other in order to determine the extent of sequence conservation among the repeats of a single rice variety. The repeats are 340 bp in length, although some of them include small deletions and/or insertions. The sequence identities vary from 79% to 94% as compared to the derived consensus sequences, named OsG340-0.
To further characterize this repetitive sequences, genomic DNA hybridization analysis was carried out using the OsG340-12 fragment as the probe. Rice total DNA was digested separately with restriction enzymes EcoRI and EcoRV because OsG340 contains no such sites. The results showed smear hybridization patterns in both digested DNAs (data not shown), indicating that OsG340 is a highly interspersed repetitive sequence in the genome.
We then explored the genome organization of the OsG340 and OsG756 sequences by PFGE. Rice chromosomal DNA larger than 6 mb was prepared from Taipei 309 and digested with rare-cutting restriction enzymes, NarI, MluI, and SmaI. These enzymes have at least one CG dinucleotide in their recognition sites and are all methylation sensitive. The digestions of rice chromosomal DNA using these enzymes yielded fragments of 150 kb to 300 kb in size. These sizes were much larger than expected based on the length of their recognition sequences. The digested fragments were separated by PFGE, and subsequent Southern blots were probed with different rice repetitive elements, as well as with the telomeric sequences of A. thaliana which has been succfessfully used as a heterologous probe to detect rice telomeres (Wu et al. 1994).
In Fig. 2, OsG340 and OsG756 were analyzed along with Os48 and the A. thaliana telomere. In Fig. 2A and B, hybridization signals with either the pOs48 or the telomere probe were observed as distinct bands, and most of these hybridization bands were common to both probes even though the relative intensities of the bands are different. This confirmed our previous conclusions that pOs48 is a tandem repeat sequence and that it is associated with the ends of several chromosomes.
The result showed the hybridization pattern of OsG340 as a long smear within a size range of 150 kb to 300 kb and several additional bands of 300 kb to 500 kb (Fig. 2C), indicating that it is an interspersed repeat. It may be present at many locations of the genome, and this is different from Os48.
OsG756 repeats were found in the centromeric, but not in the telomeric regions, by in situ hybridization. Our PFGE result showed that OsG756 is also a tandem repeat because hybridization signals appeared as distinct bands (Fig. 2D). However, their locations were different from those of Os48 and the telomere sequences because the hybridization pattern of OsG756 (Fig. 2D) was clearly unlike those of either the Os48 (Fig. 2B) or the telomere sequence (Fig. 2A).
Fig. 2. Hybridization of the filter from pulsed-field gel. Chromosomal DNA
digested with restriction enzymes, as indicated, was separated by PFGE,
transferred to a Nytran filter, and then hybridized to alpha-32P-labeled
probe: (A) A. thaliana probe, (B) pOs48 probe, (C) pOsG340 probe, and (D)
pOsG756. The DNA sequence of pOsG756 was published in Chromosoma 100, 330-338
(1991), and the accession number is L36558 for this sequence.
Only a couple hybridized bands ranging from 200-450 kb were found common to both OsG756 and Os48 probes.
The results suggest that the OsG340 and OsG756 repetitive sequences are only occasionally associated with the Os48 sequences at the ends of chromosomes. The majority of these repeats, however, are located in other places of the genome. For instance, centromeric heterochromatic regions could be the main location for the OsG756 repeats, as shown by in situ hybridization. Therefore, we believe that the genome distribution of OsG340 and OsG756 sequences differs from that of previously characterized rice repetitive elements which are predominantly located at subtelomeric regions.
We would like to thank Tiyun Wu for providing the pOsG756 clone, Esther Kahn for help in isolating OsG340 sequences, and Cathy Coster for reading the manuscript.
References
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