Hisako Ohtsubo

Isolation and characterization of IS elements repeated in the bacterial chromosome.

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.

Identification and characterization of novel retrotransposons of the gypsy type in rice.

Abstract We found that two DNA fragments, which were obtained from Oryza sativa L cv. IR36 by PCR using degenerate primers designed for amplification of a rice gene, showed homology with the rt gene encoding reverse transcriptase of the Drosophila retrotransposon gypsy. We named the element from which they originated RIRE3 (for rice retrotransposon No. 3) and analyzed it further by isolating various clones containing segments of RIRE3. Nucleotide sequencing of the clones revealed that RIRE3 has LTRs (2316 bp) and that the internal sequence (5775 bp) includes a large ORF with gag and pol regions; the pol region includes the rt gene as well as the int gene encoding integrase in this order, as in gypsy. Interestingly, the region upstream of gag in RIRE3 contained another open reading frame, here called orf0, which does not exist in gypsy or in other retrotransposons related to it. In the course of characterizing RIRE3, we obtained a further clone, which showed less homology with the pol region of RIRE3. This clone was found to be derived from another gypsy-type retrotransposon (named RIRE8) containing the LTR sequence and orf0 both of which were only weakly homologous to that in RIRE3. Further characterization of RIRE8 revealed that there were actually two subtypes of RIRE8 (named RIRE8A and RIRE8B), which show little homology to each other in the orf0 region. Although the LTRs of RIRE3 and RIRE8 elements show very weak homology with each other, there exists a conserved sequence at their termini. We therefore carried out PCR using primers which hybridize to the rt gene of RIRE3, and total genomic DNA from various monocot and dicot plants as templates, and found that a family of RIRE3 elements was present in all plants tested.

Non-LTR retrotransposons (LINEs) as ubiquitous components of plant genomes

Abstract During the course of work aimed at isolating a rice gene from Oryza australiensis by PCR, the oligonucleotide primers used were found to generate a fragment that showed sequence homology to the endonuclease (EN) region of the maize non-LTR retrotransposon (LINE) Cin4. We carried out further PCRs using oligonucleotide primers that hybridized to these sequences, and found that they amplified several fragments, each with homology to the EN regions, from Oryza sativa cv. Nipponbare as well as O. australiensis. We mapped the approximate locations of two rice LINE homologues by screening clones in a YAC library made from a rice (O. sativa) genome, and found that each homologue was present in a low copy number apparently at nonspecific regions on rice chromosomes. We then carried out PCR using degenerate oligonucleotide primers which hybridized to the rice LINE homologues and Cin4 to ascertain whether LINE homologues are present in a variety of members of the plant kingdom, including angiosperms, gymnosperms, bracken, horsetail and liverwort. Cloning and nucleotide sequencing revealed that 53 clones obtained from 27 out of 33 plant species contained LINE homologues. In addition to these homologues, we identified four homologues with EN regions in the Arabidopsis thaliana genome by a computer search of databases. The nucleotide sequences of almost all the LINE homologues were greatly diverged, but the derived amino acid sequences were well conserved, and all contained glutamic acid and tyrosine residues at almost the same relative positions as in the the active site regions of AP (apurinic/apyrimidinic)-endonucleases. The EN regions in the LINE homologues from closely related plant species show a closer phylogenetic relationship, indicating that sequence divergence during vertical transmission has been a major influence upon the evolution of plant LINEs.

PHYSICAL MAPPING OF THE 5S RIBOSOMAL RNA GENES ON RICE CHROMOSOME 11

One 5S ribosomal RNA gene (5S rDNA) locus was localized on chromosome 11 of japonica rice by in situ hybridization. The biotinylated DNA probe used was prepared by direct cloning and direct labeling methods, and the locus was localized to the proximal region of the short arm of chromosome 11 (llpl.l) by imaging methods. The distance between the signal site and the centromere is 4.0 arbitrary units, where the total length of the short arm is 43.3 units. The 5S rDNA locus physically identified and mapped in rice was designated as 5SRrn. The position of the 5S rDNA locus reported here differs from that in indica rice; possible reasons for this difference are discussed. DNA sequences of 5S rDNA are also reported.

Involvement of transposition in dispersion of tandem repeat sequences (TrsA) in rice genomes

We describe a method to identify and characterize DNA fragments containing the junction of AA genome-specific tandem repeat sequences (here called TrsA) with adjacent chromosomal sequences of rice by the polymerase chain reaction (PCR) using a pair of primers that hybridize with TrsAs and a flanking non-TrsA sequence. With this method, we obtained results suggesting that TrsA sequences present at two loci (here called trsA1 and trsA2) are flanked by direct repeats of chromosomal sequences of 172 by and about 440 by in length, respectively. These results support the idea that the TrsA sequences have been inserted into each locus by transposition, resulting in duplication of the chromosomal sequence used as target. We also describe a method to identify and characterize TrsA sequences repeated in only a few copies in the rice genome by PCR, using a pair of primers that hybridize with two different portions in the TrsA sequence, and demonstrate that TrsA sequences are present not only in rice strains with the AA genome, but also in those with non-AA genomes. The TrsA sequences were present at the trsA1 locus in all the rice strains examined, indicating that TrsA was inserted and amplified at the locus before the divergence of the various species of rice in the Oryza genus. TrsA sequences were present at the trsA2 locus, however, only in an O. sativa IR36 strain, indicating that TrsA was inserted and amplified at this locus during divergence of rice strains with the AA genome.

Transposition of Tnr1 in rice genomes to 5′-PuTAPy-3′ sites, duplicating the TA sequence

Tnr1 is a repetitive sequence in rice with several features characteristic of a transposable DNA element. Its copy number was estimated to be about 3500 per haploid genome by slot-blot hybridization. We have isolated six members of Tnr1 located at different loci by PCR (polymerase chain reaction) and determined their nucleotide sequences. The Tnr1 elements were similar in size and highly homologous (about 85%) to the Tnr1 sequence identified first in the Waxy gene in Oryza glaberrima. A consensus sequence of 235 by could be derived from the nucleotide sequences of all the Tnr1 members. The consensus sequence showed that base substitutions occurred frequently in Tnr1 by transition, and that Tnr1 has terminal inverted repeat sequences of 75 bp. Almost all the chromosomal sequences that flank the Tnr1 members were 5′-PuTA-3′ and 5′-TAPy-3′, indicating that Tnr1 transposed to 5′-PuTAPy-3′ sites, duplicating the TA sequence. PCR-amplified fragments from some rice species did not contain the Tnr1 members at corresponding loci. Comparison of nucleotide sequences of the fragments with or without a Tnr1 member confirmed preferential transposition of Tnr1 to 5′-PuTAPy-3′ sites, duplicating the TA sequence. One amplified sequence suggested that imprecise excision had occurred to remove a DNA segment containing a Tnr1 member and its neighboring sequences at the Waxy locus of rice species with genome types other than AA. We also present data that may suggest that Tnr1 is a defective form of an autonomous transposable element.

Retrotransposition of a plant SINE into the wx locus during evolution of rice

A new type of plant retroposon, p-SINE1, has been found in the wx locus of rice (Oryza sativa). It has some structural characteristics similar to those of mammalian SINEs, such as members of the Alu or Bl family. In order to estimate the time at which the integration of p-SINE1 into a single locus occurred during rice evolution, we examined the distribution of two members of p-SINE1 in several species of the Oryza genus by the polymerase chain reaction (PCR). We found that one member of p-SINE1 (p-SINE1-r2) in the ninth intron of the wx+ gene was present only in two closely related species, O. sativa and O. rufipogon, and was not present in the other species carrying the AA genome within the Oryza genus. This result indicates that p-SINE1-r2 was integrated into the wx locus after O. sativa and O. rufipogon had diverged from other species with the AA genome. In contrast to p-SINE1-r2, another member (p-SINE1-rl) located in the untranslated 5′-region of the wx+ gene was present not only in all species with the AA genome but also in species with a different genome (CCDD). This result suggests that p-SINE1-rl was integrated into that position prior to the genomic divergence. Thus, it appears that each member of p-SINE1 was retroposed at a specific site at a different time during rice evolution.

Rice transposable elements are characterized by various methylation environments in the genome

BackgroundRecent studies using high-throughput methods have revealed that transposable elements (TEs) are a comprehensive target for DNA methylation. However, the relationship between TEs and their genomic environment regarding methylation still remains unclear. The rice genome contains representatives of all known TE families with different characteristics of chromosomal distribution, structure, transposition, size, and copy number. Here we studied the DNA methylation state around 12 TEs in nine genomic DNAs from cultivated rice strains and their closely related wild strains.ResultsWe employed a transposon display (TD) method to analyze the methylation environments in the genomes. The 12 TE families, consisting of four class I elements, seven class II elements, and one element of a different class, were differentially distributed in the rice chromosomes: some elements were concentrated in the centromeric or pericentromeric regions, but others were located in euchromatic regions. The TD analyses revealed that the TE families were embedded in flanking sequences with different methylation degrees. Each TE had flanking sequences with similar degrees of methylation among the nine rice strains. The class I elements tended to be present in highly methylated regions, while those of the class II elements showed widely varying degrees of methylation. In some TE families, the degrees of methylation were markedly lower than the average methylation state of the genome. In two families, dramatic changes of the methylation state occurred depending on the distance from the TE.ConclusionOur results demonstrate that the TE families in the rice genomes can be characterized by the methylation states of their surroundings. The copy number and degree of conservation of the TE family are not likely to be correlated with the degree of methylation. We discuss possible relationships between the methylation state of TEs and their surroundings. This is the first report demonstrating that TEs in the genome are associated with a particular methylation environment that is a feature of a given TE.

Identification of a gene, tir of R100, functionally homologous to the F3 gene of F in the inhibition of RP4 transfer.

SummaryWe detected a gene of R100 functionally homologous to the F3 gene of F in the inhibition of RP4 transfer. Using in vitro recombinant DNA techniques, we located the gene, designated tir, in a 0.9 kb region, 2,392–3,293 in the nucleotide sequence coordinate of R100. From the DNA sequence analysis of R100 (Ohtsubo unpublished results), a coding frame of polypeptides, whose molecular weight is estimated to be 24.1 kilodaltons (kd), was inferred to be the region tir. Furthermore, we showed that tir could not repress expression of the F3 gene.

OsRecQ1, a QDE‐3 homologue in rice, is required for RNA silencing induced by particle bombardment for inverted repeat DNA, but not for double‐stranded RNA

Based on the nucleotide sequence of QDE-3 in Neurospora crassa, which is involved in RNA silencing, rice (Oryza sativa) mutant lines disrupted by the insertion of the rice retrotransposon Tos17 were selected. Homozygous individuals from the M(1) and M(2) generations were screened and used for further analyses. The expression of the gene was not detected in leaves or calli of the mutant lines, in contrast to the wild type (WT). Induction of RNA silencing by particle bombardment was performed to investigate any effects of the OsRecQ1 gene on RNA silencing with silencing inducers of the GFP (green fluorescence protein)/GUS (beta-glucuronidase) gene in the mutant lines. The results showed that OsRecQ1 is required for RNA silencing induced by particle bombardment for inverted-repeat DNA, but not for double-stranded RNA (dsRNA). The levels of transcripts from inverted-repeat DNA were much lower in the mutant lines than those in the WT. Furthermore, no effects were observed in the accumulation of endogenous microRNAs (miR171 and miR156) and the production of the short interspersed nuclear element retroelement by small interfering RNA. On the basis of these results, we propose that OsRecQ1 may participate in the process that allows inverted repeat DNA to be transcribed into dsRNA, which can trigger RNA silencing.