Biomedical subjects

E Ohtsubo

Publications and source records attributed to E Ohtsubo.

At least 19 recordsLinked to original sources

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↗

PubMed1995

IS1-encoded proteins, InsA and the InsA-B'-InsB transframe protein (transposase): functions deduced from their DNA-binding ability.

Insertion sequence IS1 encodes a transframe protein, InsA-B'-InsB, which is produced from two out-of-phase reading frames, insA and B'-insB, by translational frameshifting at a run of adenines. Unless the frameshifting event occurs, the InsA protein is produced from IS1. We found that cells harboring a plasmid carrying an IS1 mutant with a single adenine insertion in the run of adenines contained miniplasmids. Cloning and DNA sequencing analyses of the miniplasmids revealed that they had a deletion extending from an inverted repeat (IR) at the left end of IS1. This indicates that they were generated by IS1-mediated deletion due to efficient production of the InsA-B'-InsB transframe protein that is IS1 transposase. Both the InsA protein and transposase were partially purified as a fusion protein with collagen-LacZ by LacZ-specific affinity column chromatography. The InsA* and the collagenolyzed InsA* were found to bind specifically to a 24-bp region within each of the IRs at the ends of IS1. The transposase Tnp* and the collagenolyzed Tnp* were found to bind to the sequence with or without IR, but preferentially to that with IR. The nonspecific DNA-binding ability of transposase may be involved in recognition of the target DNA, an important process of transposition of IS1. Both InsA and transposase have the IR-specific DNA binding ability and a common polypeptide segment containing the alpha-helix-turn-alpha-helix motif, supporting the previous indication that InsA competes with transposase to bind to IRs and thus becomes a transposition inhibitor. Based on the observations described in this article, we speculate that transposase of IS1 consists of at least two domains, the N-terminal half, which almost entirely overlaps InsA, and the C-terminal half, which almost entirely overlaps B'-InsB. The frameshifting event adds the latter domain to the former to give the transposase activity recognizing IRs and the target sequence to initiate the transposition reaction.

Amino Acid Sequence↗

PubMed1995

Characterization of the functional sites in the oriT region involved in DNA transfer promoted by sex factor plasmid R100.

We have previously identified three sites, named sbi, ihfA, and sbyA, specifically recognized or bound by the TraI, IHF, and TraY proteins, respectively; these sites are involved in nicking at the origin of transfer, oriT, of plasmid R100. In the region next to these sites, there exists the sbm region, which consists of four sites, sbmA, sbmB, sbmC, and sbmD; this region is specifically bound by the TraM protein, which is required for DNA transfer. Between sbmB and sbmC in this region, there exists another IHF-binding site, ihfB. The region containing all of these sites is located in the proximity of the tra region and is referred to as the oriT region. To determine whether these sites are important for DNA transfer in vivo, we constructed plasmids with various mutations in the oriT region and tested their mobilization in the presence of R100-1, a transfer-proficient mutant of R100. Plasmids with either deletions in the sbi-ihfA-sbyA region or substitution mutations introduced into each specific site in this region were mobilized at a greatly reduced frequency, showing that all of these sites are essential for DNA transfer. By binding to ihfA, IHF, which is known to bend DNA, may be involved in the formation of a complex (which may be called oriT-some) consisting of TraI, IHF, and TraY that efficiently introduces a nick at oriT. Plasmids with either deletions in the sbm-ihfB region or substitution mutations introduced into each specific site in this region were mobilized at a reduced frequency, showing that this region is also important for DNA transfer. By binding to ihfB, IHF may also be involved in the formation of another complex (which may be called the TraM-IHF complex) consisting of TraM and IHF that ensures DNA transfer with a high level of efficiency. Several-base-pair insertions into the positions between sbyA and sbmA affected the frequency of transfer in a manner dependent upon the number of base pairs, indicating that the phasing between sbyA and sbmA is important. This in turn suggests that both oriT-some and the TraM-IHF complex should be in an appropriate position spatially to facilitate DNA transfer.

Base Sequence↗

PubMed1994

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 bp 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.

Adenine Nucleotides↗

PubMed1994

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 bp and about 440 bp 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.

Base Sequence↗

PubMed1994

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 (11p1.1) 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 5SrDNA 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.

Base Sequence↗

PubMed1994

Translational control in production of transposase and in transposition of insertion sequence IS3.

IS3 (1258 bp in length) contains two open reading frames, orfA and orfB, which are out of phase and overlap each other. We show here that three proteins of 10, 32 and 42 kDa in size are encoded by IS3. The 10 kDa protein is the product of orfA and is here called OrfA. The ATG codon of orfB which overlaps the termination codon of orfA is utilized to produce the 32 kDa protein (here called OrfB), in a manner depending on translation of orfA. The 42 kDa protein is a transframe protein (here called OrfB), which is synthesized from orfA and orfB by -1 translational frameshifting at the A4G motif present in the overlapping region. Both the frameshifting event to produce OrfB and the coupled translation event to produce OrfB are greatly stimulated by a pseudo knot structure located in the overlapping region between orfA and orfB. A mutant IS3 with a single base insertion in the A4G motif efficiently produces the OrfB transframe protein without frameshifting. This mutant was found not to mediate co-integration but to mediate adjacent deletion to produce various miniplasmids and minicircles in large amounts. The OrfB transframe protein is necessary and sufficient for formation of these deletion products, implying that it is the transposase. Most of the minicircles consisted solely of the entire IS3 sequence and a three base-pair sequence between the IS3 ends. The significance of minicircle formation is discussed.

Amino Acid Sequence↗

PubMed1994

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 B1 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-r1) 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-r1 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.

Base Sequence↗

PubMed1994

Site- and strand-specific nicking at oriT of plasmid R100 in a purified system: enhancement of the nicking activity of TraI (helicase I) with TraY and IHF.

We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, TraI and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the TraI protein and Mg2+. The products were complex DNA molecules with a protein covalently linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the TraI protein alone, indicating that the protein attached at the 5' end of the nick is the TraI protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the TraI and TraY proteins at oriT.

Bacterial Proteins↗

PubMed1994

Mapping and disruption of the chpB locus in Escherichia coli.

The chpB locus is a chromosomal homolog of the pem locus, which is responsible for stable maintenance of plasmid R100 within the host cells. Like pem, chpB codes for two genes, chpBK and chpBI, encoding a growth inhibitor and a suppressor for the killing action of the ChpBK protein, respectively. Here, we determined the precise location of the chpB locus, which is linked to ileR and ppa in the order ileR-chpB-ppa, at 95.7 min on the map of Escherichia coli. We then constructed mutants with an insertion of a (cat) fragment within chpBK or chpBI on the E. coli chromosome. These mutants grew normally, indicating that chpB is dispensable for cell growth.

Base Sequence↗

PubMed1994

Identification of the region that determines the specificity of binding of the transposases encoded by Tn3 and gamma delta to the terminal inverted repeat sequences.

To analyze the region that determines the specificity of binding of the Tn3 transposase to the terminal inverted repeat sequences (IR), we first determined the nucleotide sequence of a Tn3-family transposon, gamma delta, which is supposed to encode a transposase similar to that of Tn3. gamma delta was 5981 bp in length and contained three coding frames: Two were the genes, tnpA and tnpR, encoding transposase (1002 amino acids) and resolvase/repressor (183 amino acids), respectively, and the third, named tnpX, encoding a protein (698 amino acids) of unknown function but containing two NTP-binding motifs. Utilizing the tnpA sequence, we then constructed a series of Tn3-gamma delta hybrid genes encoding chimeric proteins in the N-terminal segments of the transposases (amino acid position 1 to 242 of Tn3 or 1' to 243' of gamma delta), which has been previously shown to be responsible for specific binding of transposase to IR sequences in Tn3. Examination of their DNA-binding activities revealed that the subsegment of the N-terminus from amino acid position 1 to 109 determines the specificity of binding to the IR sequences. The third coding frame found in gamma delta, tnpX, is located downstream of tnpR and is expressed from the tnpR promoter in the absence of the tnpR gene product, resolvase/repressor, to produce a protein that inhibits the growth of the host cells. Possible roles of this protein are discussed.

Amino Acid Sequence↗

PubMed1993

Distribution of the Shigella sonnei insertion elements in Enterobacteriaceae.

The distribution of IS1, IS600, IS629, IS630 and IS640, present in an Shigella sonnei strain, was examined in strains belonging to various species of enteric bacteria. Four Shigella species including Sh. sonnei contained all IS elements, several of which were in large numbers, and showed species-specific distribution patterns. The other strains contained some of the IS elements in a few copies or none at all, except for some clinical isolates in the Escherichia coli strains, which showed similar distribution patterns to those of the Shigella species, suggesting that the E. coli isolates are closely related to those in Shigella. The IS elements examined may be used to classify various bacterial strains and to identify the Shigella strains and some of the E. coli strains to be isolated from various sources.

DNA Transposable Elements↗

PubMed1993

DNA binding domains in Tn3 transposase.

Various segments of Tn3 transposase were fused individually to beta-galactosidase, and the resulting fusion proteins were examined for their DNA binding ability by a nitrocellulose filter binding assay. Analyses of a series of the fusion proteins revealed that the N-terminal segment of the transposase (amino acid positions 1-242; the transposase gene encodes 1004 residues in all) had specific DNA binding ability for the 38 bp terminal inverted repeat (IR) sequence, and the central segment (amino acid positions 243-632) had non-specific DNA binding ability. Further analyses of each of the two regions revealed that the N-terminal segment could be divided into at least two subsegments (amino acid positions 1-86 and 87-242), neither of which had specific DNA binding ability, but which both possessed non-specific DNA binding ability. The central segment included two subsegments (amino acid positions 243-289 and 439-505) with non-specific DNA binding ability. These results and other observations suggest that Tn3 transposase has several domains including those responsible for non-specific DNA binding, and a combination of two or more domains gives rise to specific DNA binding activity. The C-terminal segment of the transposase (amino acid positions 633-1004), which is very well conserved among transposases encoded by Tn3 family transposons, had no DNA binding ability. This segment may represent the main part of the catalytic domain responsible for the initiation step of transposition.

Amino Acid Sequence↗

PubMed1993

Repression of the traM gene of plasmid R100 by its own product and integration host factor at one of the two promoters.

Plasmid R100 codes for the traM gene, which is required for DNA transfer and whose product has been shown to bind to the four sites, called sbmA to sbmD, upstream of traM. To determine whether the TraM protein regulates the expression of traM, we constructed the plasmids carrying various portions of the region upstream of the initiation codon ATG for traM, which was fused with lacZ in frame, and introduced them into the cells, which did or did not harbor another compatible plasmid carrying traM. We then assayed the beta-galactosidase (LacZ) activity to monitor the expression of the fusion genes and analyzed the traM-specific transcripts made in the cells. Two promoters for traM were identified and designated pM1 and pM2. Promoter pM2 lies upstream of pM1 and overlaps the sbmC-sbmD region. Promoter pM1 is constitutively expressed, while pM2 is much stronger but is repressed almost completely by the TraM protein and partially by integration host factor, whose binding site is near pM2. The traM gene is likely to be expressed from pM2 when the TraM protein is at low levels after dilution in the donor cell during cell growth or before its expression in the recipient cell which has just received R100 by conjugation. The expression from pM2 could maintain the amount of the TraM protein at a constant level needed to initiate DNA transfer at any time. Integration host factor, which can partially repress the traM gene, may play a role in forming an active complex with the TraM protein at the sbm region to facilitate DNA transfer.

Amino Acid Sequence↗

PubMed1993

chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100.

The pem locus is responsible for stable maintenance of plasmid R100 and consists of two genes, pemI and pemK. The pemK gene product is a growth inhibitor, while the pemI gene product is a suppressor of this inhibitory function. We found that the PemI amino acid sequence is homologous to two open reading frames from Escherichia coli called mazE and orf-83, which are located at 60 and 100 min on the chromosome, respectively. We cloned and sequenced these loci and found additional open reading frames, one downstream of each pemI homolog, both of which encode proteins homologous to PemK. The pem locus homolog at 60 min was named chpA and consists of two genes, chpAI and chpAK; the other, at 100 min, was named chpB and consists of two genes, chpBI and chpBK. The distal portion of chpBK was found to be adjacent to the ppa gene that encodes pyrophosphatase, whose map position had not been previously determined. We then demonstrated that the chpAK and chpBK genes encode growth inhibitors, while the chpAI and chpBI genes encode suppressors for the inhibitory function of the ChpAK and ChpBK proteins, respectively. These E. coli pem locus homologs may be involved in regulation of cell growth.

Amino Acid Sequence↗

PubMed1993

Classification and relationships of rice strains with AA genome by identification of transposable elements at nine loci.

We analyzed the presence of p-SINE1 members at five loci in the rice strains belonging to seven species with AA genome in the Oryza genus by the methods including polymerase chain reaction (PCR). Four p-SINE1 members (p-SINE1-r3, r4, r5 and r7) were present at the corresponding loci in all the strains examined. One member (p-SINE1-r6) was, however, not present at the corresponding locus in most of the African strains of O. glaberrima and O. barthii, but was in the other strains. The PCR-amplified fragments containing p-SINE1-r4 in many strains were found to be larger due to insertion of either one of two transposable elements, named Tnr2 and Ret1, within or near p-SINE1-r4, respectively: Tnr2 is 157 bp in length with terminal inverted repeat sequences of about 56 bp; Ret1 is only 13 bp in length with a T stretch at its end. Tnr2 was not present in the corresponding locus in all the strains belonging to O. sativa Japonica and in some strains of O. rufipogon and O. longistaminata, while Ret1 was present only in the two strains of O. longistaminata. These results and previous ones obtained from the analysis of the other two p-SINE1 members (p-SINE1-r1 and r2) in the Wx gene indicate that the elements, such as p-SINE1-r6, Tnr2, Ret1 and p-SINE1-r2, have been inserted into the respective loci during divergence of the rice species with AA genome. The patterns for the presence and absence of the transposable elements at the respective loci enabled us to classify the rice strains with AA genome into ten groups and to infer their relationships.

Base Sequence↗

PubMed1992

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.

Amino Acid Sequence↗