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E Ohtsubo

Publications and source records attributed to E Ohtsubo.

At least 19 recordsLinked to original sources
PubMed1999

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

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.

Amino Acid Sequence↗
PubMed1998

Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157:H7 derived from Sakai outbreak.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, derived from an outbreak in Sakai city, Japan in 1996, possesses two kinds of plasmids: a 93-kb plasmid termed pO157, found in clinical EHEC isolates world-wide and a 3.3-kb plasmid termed pOSAK1, prevalent in EHEC strains isolated in Japan. Complete nucleotide sequences of both plasmids have been determined, and the putative functions of the encoded proteins and the cis-acting DNA sequences have been analyzed. pO157 shares strikingly similar genes and DNA sequences with F-factor and the transmissible drug-resistant plasmid R100 for DNA replication, copy number control, plasmid segregation, conjugative functions and stable maintenance in the host, although it is defective in DNA transfer by conjugation due to the truncation and deletion of the required genes and DNA sequences. In addition, it encodes several proteins implicated in EHEC pathogenicity such as an EHEC hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP) and type II secretion system. pOSAK1 possesses a ColE1-like replication system, and the DNA sequence is extremely similar to that of a drug-resistant plasmid, NTP16, derived from Salmonella typhimurium except that it lacks drug resistance transposons.

DNA, Bacterial↗
PubMed1998

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Alleles↗
PubMed1998

Regulatory mechanisms in expression of the traY-I operon of sex factor plasmid R100: involvement of traJ and traY gene products.

BACKGROUND: The plasmid R100 encodes tra genes essential for conjugal DNA transfer in Escherichia coli. Genetic evidence suggests that the traJ gene encodes a positive regulator for the traY-I operon, which includes almost all the tra genes located downstream of traJ. The molecular mechanism of regulation by TraJ, however, is not yet understood. traY is the most proximal gene in the traY-I operon. TraY promotes DNA transfer by binding to a site, sbyA, near the origin of transfer. TraY is suggested to have another role in regulation of the traY-I operon, since it binds to two other sites, named sbyB and sbyC, located in the region preceding traY-I. RESULTS: Using a traY-lacZ fusion gene, we showed that the traY-I operon was expressed only in the presence of traJ. The TraJ-dependent expression of traY-I required the E. coli arcA gene, which encodes a host factor required for conjugation. TraJ-dependent transcription occurred from a promoter (named pY) located upstream of traY-I. The isolated TraJ protein was found to bind to a dyad symmetry sequence, named sbj (specific binding site of TraJ), which existed in the intergenic region between traJ and traY-I. We also demonstrated that TraY repressed the TraJ-dependent expression of traY-I at the TraY binding sites, sbyB and sbyC, which overlapped with pY. CONCLUSIONS: TraJ is a protein which binds to the sbj site in the region upstream of the promoter pY and positively regulates expression of the traY-I operon in the presence of the E. coli arcA gene. Since sbj is located 93bp upstream of pY in the intergenic region between traJ and traY-I, TraJ presumably contacts with a transcription apparatus to promote transcription from pY. TraY, which is known to activate the initiation of conjugal DNA transfer, has a new role in the transcriptional autoregulation of traY-I expression. At levels which are sufficient to initiate conjugal DNA transfer, TraY represses traY-I transcription in the presence of TraJ.

Bacterial Outer Membrane Proteins↗
PubMed1997

Isolation and characterization of IS1 circles.

Transposase encoded by insertion sequence IS1 is produced from two out-of-phase reading frames by translational frameshifting that occurs in a run of adenines. An IS1 mutant with a single adenine insertion in the run of adenines efficiently produces transposase, resulting in generation of miniplasmids by deletion for a region adjacent to IS1 from a plasmid carrying the IS1 mutant. Here, we found that besides miniplasmids, cells harboring the plasmid contained minicircles without the region required for replication. Cloning and DNA sequencing of the minicircles revealed that most of them were IS1 circles consisting of the entire IS1 sequence and a sequence, 5-9 bp in length, which intervenes between terminal inverted repeats, IRL and IRR, of IS1. Analysis of more IS1 circles isolated by polymerase chain reaction revealed that the intervening sequence was derived from the region flanking either IRL or IRR in the parental plasmid, suggesting that IS1 circles are generated by an excision event from the parental plasmid. The IS1 circles may be formed due to the cointegration reaction occurring within the parental plasmid carrying IS1.

Cloning, Molecular↗
PubMed1997

Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza.

Large variation in genome size as determined by the nuclear DNA content and the mitotic chromosome size among diploid rice species is revealed using flow cytometry and image analyses. Both the total chromosomal length (r = 0.939) and the total chromosomal area (r = 0.927) correlated well with the nuclear DNA content. Among all the species examined, Oryza australiensis (E genome) and O. brachyantha (F genome), respectively, were the largest and smallest in genome size. O. sativa (A genome) involving all the cultivated species showed the intermediate genome size between them. The distribution patterns of genome-specific repetitive DNA sequences were physically determined using fluorescence in situ hybridization (FISH). O. brachyantha had limited sites of the repetitive DNA sequences specific to the F genome. O. australiensis showed overall amplification of genome-specific DNA sequences throughout the chromosomes. The amplification of the repetitive DNA sequences causes the variation in the chromosome morphology and thus the genome size among diploid species in the genus Oryza.

Base Sequence↗
PubMed1997

Inhibition of transpositional recombination by OrfA and OrfB proteins encoded by insertion sequence IS3.

BACKGROUND: An insertion element IS3 is flanked by terminal inverted repeat (IR) sequences. IS3 encodes two, out-of-phase, overlapping open reading frames, orfA and orfB, from which three proteins are produced. OrfAB is a transframe protein produced by -1 translational frameshifting between orfA and orfB, and it is known to be IS3 transposase. OrfA and OrfB are the proteins produced without frameshifting, but their functions have not been elucidated. RESULTS: A plasmid carrying an IS3 mutant that produces only transposase generates miniplasmids--which are the IS3-mediated intramolecular transposition products--as well as characteristic IS3 circles and linear IS3 molecules. OrfA inhibited the generation of these small molecules to a lesser degree, but OrfB did not. OrfB, together with OrfA, however, inhibited the generation more strongly than OrfA alone. OrfA also inhibited the intermolecular transposition of mini-IS3 with the chloramphenicol-resistance gene flanked by IRs to a reduced frequency, and OrfB together with OrfA inhibited it almost completely. OrfA and/or OrfB did not, however, repress transcription from the promoter in the left-terminal region preceding orfA. CONCLUSIONS: The results obtained above show that OrfA and OrfB are not repressors but are inhibitors of transpositional recombination promoted by transposase. OrfA with an alpha helix-turn-alpha helix DNA-binding motif may compete with transposase to bind to terminal IRs. OrfA, together with OrfB that has a DDE motif conserved in retroviral integrases, may inhibit the formation of an active transpososome consisting oftransposase, two terminal IRs and target DNA for the strand transfer reaction. IS3 with a limited size, 1258 bp in length, uses strategies of translational frameshifting and coupling to produce transposase as well as negative regulators to make its copies at a low level, which minimizes a deleterious effect of transposition on bacterial hosts.

Bacterial Proteins↗
PubMed1997

Roles of TraI protein with activities of cleaving and rejoining the single-stranded DNA in both initiation and termination of conjugal DNA transfer.

BACKGROUND: The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood. RESULTS: We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region. CONCLUSIONS: Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.

Base Sequence↗
PubMed1997

RIRE1, a retrotransposon from wild rice Oryza australiensis.

RIRE1 is a retrotransposon present in wild rice Oryza australiensis in an extraordinary number of copies, and only a portion of the LTR sequence has been determined previously. Here, we isolated and sequenced DNA segments of various portions of RIRE1, revealing that the sequences of LTR and the internal region were 1523 and 5277 bp in length, respectively. The internal region shows homology with the pol region in copia, a Drosophila retrotransposon, indicating that RIRE1 is a copia-like retrotransposon. The internal region of RIRE1 contained an open reading frame coding for genes, gag, pro, int, rt and rh, like copia and retroelements related to it. A clone screened from a library of the O. australiensis genomic DNA contained solo LTR, which was flanked by direct repeats of a 5-bp sequence. This suggests that RIRE1 generates a duplication of the target sequence of 5 bp upon retroposition. We observed that many RIRE1 members were nested by another RIRE1 member. This indicates that these RIRE1 members have received another RIRE1 to make an extraordinary number of copies in the O. australiensis genome without giving a deleterious effect on the growth of rice cells.

Amino Acid Sequence↗
PubMed1996

Identification of Tnr3, a suppressor-mutator/enhancer-like transposable element from rice.

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion.Aa 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1539 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5'-CACTA---3'; these repeats are similar to those found in members of the En/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to the En/Spm family and is thus named Tnr3 (transposable element in rice no. 3). In fact, Tnr carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of the En/Spm family. The subterminal repeat sequences in Tnr3 are, however, of two kinds, although they share homology with each other. Tnr3 and its relatives were present in multiple copies in rice. considering the length of Tnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.

Base Sequence↗
PubMed1996

Identification and characterization of the linear IS3 molecules generated by staggered breaks.

Insertion sequences IS3 encodes two, out-of-phase, overlapping open reading frames, orfA and orfB. The OrfAB transframe protein that is IS3 transposase is produced by -1 translational frameshifting between orfA and orfB. Efficient production of the IS3 transposase in the cells harboring the IS3-carrying plasmid has been shown to generate miniplasmids as well as characteristic minicircles, called IS3 circles, consisting of the entire IS3 sequence and one of the 3-base pair sequences flanking IS3 in the parental plasmid. Here, we show that the IS3 transposase also generates the linear molecules of IS3 with 3-nucleotide overhangs at the 5'-ends. The nucleotide sequences of the overhangs are the same as those flanking IS3 in the parental plasmid, suggesting that the linear IS3 molecules are generated from the parental plasmid DNA by staggered double strand breaks at the end regions of IS3. The linear IS3 molecules are likely to be the early intermediates in the transposition reaction, which proceeds in a non-replicative manner.

Base Sequence↗
PubMed1996

A cell-free system of Tn3 transposition and transposition immunity.

BACKGROUND: Tn3 is a bacterial transposon, which encodes transposase required for its transposition. Tn3 has terminal inverted repeat (IR) sequences of 38 bp in length, whose inner region, called the B domain, is bound by transposase. Tn3 confers transposition immunity, a phenomenon in which Tn3 transposes to a target replicon with Tn3 much less frequently than to a target replicon with no Tn3. RESULTS: To understand transposition and transposition immunity at the molecular level, we constructed a cell-free system using a plasmid as the target. Transpositional recombination occurred in a cell extract containing transposase between the target and a donor plasmid carrying mini-Tn3 at a high frequency. The reaction required ATP, Mg2+, dNTPs and 2% polyvinyl alcohol, and was inhibited by inhibitors for DNA synthesis and DNA gyrase. In this system, when a plasmid with the IR sequence was used as the target, the frequency of transposition was significantly decreased, demonstrating that the transposition immunity conferred by Tn3 is reproduced in vitro. Preincubation of the target in the cell extract increased the level of transposition immunity. On the other hand, mutations within the B domain in the IR sequence of the target abolished transposition immunity. CONCLUSIONS: Transposition of Tn3 and transposition immunity could be reproduced in vitro. The results demonstrate that the binding of transposase to domain B of the IR sequence in the target replicon is responsible for transposition immunity. We propose that the transposition immunity results from conversion of the normal synaptic complex formed between the donor and target molecules to another complex which is inactive for transposition, due to the interaction between transposases binding to the IR sequences in the donor and target molecules.

Base Sequence↗
PubMed1996

Specific nicking at the 3' ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP.

BACKGROUND: Tn3, a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition. The transposition of Tn3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini-Tn3 as the donor and another plasmid as the target. Transposase has the ability to bind to the 38-bp terminal inverted repeats (IRs) of Tn3. The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood. RESULTS: We found that nicking occurred efficiently in the cell-free system at each of the 3' ends of the IRs of mini-Tn3 in the closed circular or linear donor molecules. The nicking reaction required transposase and Mg2+, but did not require ATP, an ATP-regenerating system, dNTPs and polyvinyl alcohol, which were the requirements for the transposition reaction. By using the nicking assay employed here, transposase was purified almost to homogeneity. Gel filtration and sedimentation analyses indicate that transposase forms a dimer in a solution containing 0.5 M NaCl. The nicking activity of the purified transposase was weak and was found to be stimulated by a host factor. The nicking stimulation factor was subsequently purified and found to be ACP, an Escherichia coli acyl carrier protein. CONCLUSIONS: Nicking occurred efficiently at the 3' ends of mini-Tn3 in the reaction mixture containing transposase and ACP. ACP is known to act as a factor which modulates enzymes that are involved in several biological processes either in the acylated or unacylated form. ACP may also modulate transposase to initiate the transposition reaction with nicking at the 3' ends of Tn3.

Acyl Carrier Protein↗
PubMed1996

Characterization of poly-leucine substituted analogues of the human surfactant protein SP-C.

A series of novel amphipathic peptides constituted of an N-terminal hydrophilic portion (CPVHLKR, residues 6-12) of human pulmonary surfactant protein-C (SP-C) and a poly-leucine (poly-L) stretch of various chain lengths as the C-terminal hydrophobic tail were synthesized and evaluated relevant to their ability to improve the surface activity of a ternary lipid mixture composed of dipalmitoylphosphatidylcholine, egg-phosphatidylglycerol and palmitic acid (DPPC/E-PG/PA, 75:25:10, w/w) in a Langmuir-Wilhelmy surface balance. CPVHLKRL11, a human SP-C analogue bearing an 11-residue poly-L tail, and its related peptides with longer tails in the ternary lipid mixture, accelerated not only the surface spreading at the air-water interface but also exhibited significantly improved dynamic surface activity, compared to the ternary lipid mixture. Their surface activities were almost indiscernible from those of the synthetic human SP-C. When reconstituted into a ternary lipid mixture containing members of the homologous series of n-saturated diacylphosphatidylglycerol, the surface activities of the poly-L analogues were almost completely unaffected, whereas replica peptides carrying the hydrophobic portion of native SP-C were found to have distinct surface activities depending upon the acyl-chain lengths of phosphatidylglycerol. The poly-L stretch of a poly-L analogue could be replaced with poly-norleucine of the same chain length without a significant loss of surface activity. Substitution of the poly-L portion in the analogues with poly-valine or poly-isoleucine resulted in a considerable decrease in surface activity. The poly-L analogue in the DPPC/E-PG/PA mixture was demonstrated to act as an excellent surfactant comparable with Surfacten, a modified bovine surfactant preparation that was used for treatment for infant respiratory distress syndrome, based on evaluation of the lung pressure-volume characteristics using premature rabbit neonates.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of two tandem repeat sequences (TrsB and TrsC) and a retrotransposon (RIRE1) as genome-general sequences in rice.

Three kinds of DNA sequences (here called TrsB, TrsC and RIRE1) have been previously reported to be those repeated in tandem specifically in the wild rice species with FF, CC or EE genome, respectively. To characterize these genome type-specific sequences, we carried out PCR using a pair of primers, which hybridize to a restricted region in the repeating unit sequence and prime DNA synthesis in both directions. Gel electrophoresis and DNA sequencing revealed that PCR using primers for TrsB (or TrsC) amplified the fragments with an integral series of a unit length not only from total DNA of the rice strain with FF (or CC) genome, but also from those of the rice strains with non-FF (or non-CC) genome. TrsB or TrsC was, however, found to be repeated in an extraordinary number of copies in the species with FF or CC genome, respectively, in which the TrsB (or TrsC) sequence has been originally identified. PCR using primers for RIRE1 produced various sizes of fragments from total DNA of the rice strains with EE genome. The fragments, however, showed no progression at interval of the unit length characteristic for tandem repeats. Nucleotide sequencing of the amplified fragments revealed that they were not the sequences repeated in tandem, but were those interspersed as an element having partial homology with the LTR sequences of retrotransposons, Wis-2-1A in wheat and BARE-1 in barley. RIRE1 was present in the rice species with any types of genomes, but in the species with EE genome in an extraordinary number of copies.

Base Sequence↗
PubMed1995

Energetics of coupled twist and writhe changes in closed circular pSM1 DNA.

The extent of local denaturation in closed circular pSM1 DNA depends upon the linking difference, delta Lk, and the temperature, t. We have determined the denaturation profiles, using gel electrophoresis, over the ranges -37 < or = delta Lk < or = +16 and 25 degrees C < or = t < or = 65 degrees C. We have applied statistical mechanical methods to these data to evaluate the free energies of superhelix formation, of the twisting of single strands around each other, and of the initration of local denaturation. Because the complete nucleotide sequence is needed for this analysis, the complete pSM1 DNA sequence was determined and is reported here. The values of the free energy parameters found in this work agree closely with those previously obtained from experiments with pBR322 DNA, suggesting that there is little dependence of these values on the particular DNA sequence. We find the temperature dependence of these free energies by the appropriate statistical mechanical analysis of the temperature-dependent denaturation profiles produced by supercoiling. Calculations of the transition probability profiles indicate that the course of local denaturation in pSM1 DNA involves a complex competition among several sites of comparable susceptibility. This contrasts with the melting of pBR322 DNA, in which one principal site dominates. In both molecules the sites of predicted denaturation occur at or near regulatory regions, suggesting that duplex destabilization may be associated with their biological activities.

Base Sequence↗
PubMed1995

Large scale purification and characterization of TraI endonuclease encoded by sex factor plasmid R100.

The TraI protein encoded by plasmid R100 was purified in a large scale by monitoring the strand- and site-specific nicking activity at the origin of transfer, oriT. The N-terminal amino acid sequence of the purified protein was identical to that deduced from the DNA sequence of an open reading frame encoding TraI. The TraI protein is a DNA helicase which is highly processive and unwinds DNA in the 5' to 3' direction. The Stokes radius and the sedimentation coefficient for the TraI protein in 200 mM NaCl indicate that the protein is a rod-shaped monomer, whose native molecular weight is 186,000. Chemical cross-linking analysis revealed that there exist more dimers of TraI under the low salt conditions, under which both nicking and unwinding reactions catalyzed by TraI are the most efficient, indicating that the TraI protein is functionally active in a dimer form. TraI hardly introduced a nick into the linearized plasmid DNA and only slightly into the relaxed closed circular DNA, indicating that TraI requires superhelical structure of substrate DNA for the nicking reaction. Deletion analysis in the oriT region revealed that a particular region of 54 base pairs containing oriT is required for the nicking reaction.

Amino Acid Sequence↗