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E Ohtsubo

Publications and source records attributed to E Ohtsubo.

At least 91 records · Page 5Linked to original sources
PubMed1990

Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100.

The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.

Amino Acid Sequence↗
PubMed1990

Tn3 transposition immunity is conferred by the transposase-binding domain in the terminal inverted-repeat sequence of Tn3.

A series of mutant terminal inverted repeats (IRs), having 2 bp substitutions at various sites within the 38-bp IR sequence of the ampicillin-resistance transposon Tn3, were tested for transposition immunity to Tn3. Mutations within region 1-10 in the IR did not affect transposition immunity, while mutations within region 13-38 inactivated the immunity function. These two regions corresponded to domain A which was not bound specifically by Tn3 transposase and to domain B which was bound by the transposase, respectively. This indicates that specific binding of transposase to domain B within the IR sequence is responsible for transposition immunity.

Ampicillin Resistance↗
PubMed1990

Two domains in the terminal inverted-repeat sequence of transposon Tn3.

Tn3 and related transposons have terminal inverted repeats (IR) of about 38 bp that are needed as sites for transposition. We made mini-Tn3 derivatives which had a wild-type IR of Tn3 at one end and either the divergent IR of the Tn3-related transposon, gamma delta or IS101, or a mutant IR of Tn3 at the other end. We then examined both in vivo transposition (cointegration between transposition donor and target molecules) of these mini-Tn3 elements and in vitro binding of Tn3-encoded transposase to their IRs. None of the elements with an IR of gamma delta or IS101 mediated cointegration efficiently. This was due to inefficient binding of transposase to these IR. Most mutant IR also interfered with cointegration, even though transposase bound to some mutant IR as efficiently as it did to wild type. This permitted the Tn3 IR sequence to be divided into two domains, named A and B, with respect to transposase binding. Domain B, at positions 13-38, was involved in transposase binding, whereas domain A, at positions 1-10, was not. The A domain may contain the sequence recognized by some other (e.g., host) factor(s) to precede the actual cointegration event.

Base Sequence↗
PubMed1990

Mapping of insertion element IS30 in the Escherichia coli K12 chromosome.

We identified seven phage clones containing the insertion element IS30 in a lambda phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K12 W3110 (Kohara et al. 1987). We could assign locations and orientations to four copies of IS30 (named is30A to is30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them. These IS30s were present at the same locations in chromosomes of both W3110 and another E. coli K12 strain JE5519, and thus are assumed to be present in other E. coli K12 derivatives, including early isolates. Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 bp stretch of the right terminal region of IS30.

Base Sequence↗
PubMed1990

Site-specific transposition of insertion sequence IS630.

IS630 is a 1.15-kilobase sequence in Shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. To assess its transposition, we constructed composite elements containing inverted copies of IS630 flanking a drug resistance gene. We found that these composite elements transposed to plasmid ColE1 in Escherichia coli. DNA sequencing showed that transposition was, in all cases, to the dinucleotide sequence 5'-TA-3'. There were two preferred insertion sites which corresponded to the TA sequences in the inverted repeats of a 13-base-pair stem region of the [rho]-dependent transcription terminator. IS630 is flanked by TA, and nucleotide substitution by in vitro mutagenesis at these ends did not affect transposition activity of a composite element or its ability to insert preferentially into TA within the 13-base-pair inverted repeat sequences or to duplicate the target sequence.

Base Composition↗
PubMed1989

Mapping of insertion elements IS1, IS2 and IS3 on the Escherichia coli K-12 chromosome. Role of the insertion elements in formation of Hfrs and F' factors and in rearrangement of bacterial chromosomes.

The chromosome of an Escherichia coli K-12 strain W3110 contains seven copies of insertion element IS1, 12 copies of IS2 and six copies of IS3. We determined the approximate locations of six copies of IS1 (named is1A to is1F), ten copies of IS2 (named is2A to is2J), and five copies of IS3 (named is3A to is3E) on the W3110 chromosome by plaque hybridization using the "mini-set" of the lambda phage library that includes 476 clones carrying chromosomal segments that cover the W3110 chromosome almost entirely. Cleavage maps of the W3110 chromosome and cleavage analysis of phage DNAs carrying insertion elements allowed us to assign more precise locations to most of the insertion elements and to determine their orientations. Insertion elements were distributed randomly along the W3110 chromosome in one or other orientation. Several of these were located at the same positions on the chromosome of another E. coli K-12 strain, JE5519, and they were assumed to be the original complement of insertion elements in E. coli K-12 wild-type. Locations and orientations of such insertion elements were correlated well with Hfr points of origin and with crossover points for excision of some F' factors derived from several Hfrs. Insertion elements may be involved also in rearrangement of bacterial chromosomes.

Chromosomes, Bacterial↗
PubMed1989

Effect of the pem system on stable maintenance of plasmid R100 in various Escherichia coli hosts.

We cloned the pem segment of plasmid R100 containing the two genes pemI and pemK, which are responsible for stable maintenance of R100 in dividing cells, into pHS1, a temperature-sensitive replication mutant of plasmid pSC101. We then examined the effect of the pem system on the maintenance of the resultant pem+ plasmid pDOM17 in various Escherichia coli host strains upon inhibition of replication of the plasmid at a high temperature. We show that the pem+ plasmid was maintained stably in the cell population and efficiently in the two hosts, km1213 (polAts) and KP64 (recA), but less efficiently in others, such as W3110, C600, P3478 (polA), and SH2743 (sfiA sfiC); the rate of cell growth was reduced at or after the time when the copy number of pDOM17 was supposed to be 0 in all of the hosts examined. We also show that a large fraction of the non-viable pDOM17-free segregant cells was produced in the former two hosts, while a smaller fraction of such cells was produced in the latter hosts, in which cell division was inhibited for several generations. Based on these results and other observations, we point out that the pemK gene product has the function not to kill the plasmid-free segregant cells, but primarily to inhibit division of these segregants. Inhibition of cell division secondarily leads to death of the plasmid-free segregants very efficiently in the two particular hosts, resulting in an apparently more stable maintenance of the pem+ plasmid in these two hosts than in others.

Cell Division↗
PubMed1989

The oncogenicity of avian adenoviruses. IV. Confirmatory evidence for recombination between viral and cellular DNA sequences and repetition of the recombinant in cells of a tumor line.

On the basis of hybridization analyses with viral DNA fragments as probes, we previously concluded that, in DNAs of many fowl adenovirus type 1-induced rodent tumors, units consisting of viral DNA segments flanked on both sides by cellular DNA segments are repeated more than 100 times per diploid genome of cell. Molecular cloning of virus-cellular junction fragments and analyses of their sequence arrangement in the present study confirmed that the previous conclusion was basically correct.

Adenoviridae↗
PubMed1989

Frameshifting is required for production of the transposase encoded by insertion sequence 1.

Insertion sequence IS1 has two coding frames, insA and insB, which are essential for its transposition. Here, we show that a frameshifting event in the -1 direction from the 3' end region of the insA frame to an open reading frame (B' frame), extending from the 5' end of the insB frame, is involved in production of the InsA-B'-InsB fusion protein that has IS1 transposase activity. The frameshifting event is likely to have occurred at the sequence AAAAAC where the insA frame overlaps the B' frame. Interestingly, this sequence is also present in one of the two sequences identified in retroviruses as frameshift signals for production of transframe polyproteins from the overlapping genes gag-pro or gag-pro-pol.

Amino Acid Sequence↗
PubMed1988

Unidirectional replication of plasmid R100.

We isolated a 284 base-pair BamHI fragment of plasmid R100 that supports initiation of replication of a plasmid regardless of the orientation of the fragment. Analysis of the specific radioactivity of restriction fragments from 32P-labeled replication intermediates synthesized in vitro shows that replication of the plasmid carrying the 284 base-pair fragment is unidirectional. The direction of replication depends on the orientation of the fragment present in the plasmid. The 5' ends of the leading-strand DNA formed in the early stage of replication were mapped to a region downstream from the 284 base-pair fragment in the direction of replication. The lagging-strand DNA products were also identified and their 3' ends mapped to unique sites within the 284 base-pair fragment causing unidirectional replication of R100.

Autoradiography↗
PubMed1988

The oriC unwinding by dam methylation in Escherichia coli.

It has been shown that dam methylation is important in the regulation of initiation of DNA replication in E.coli. The question then arises as to whether dam methylation in the oriC region mediates any structural changes in DNA involved in the regulation of initiation of DNA replication. We demonstrate that the thermal melting temperature of the oriC region is lowered by adenine methylation at GATC sites. The regulation of initiation of DNA replication by dam methylation may be attributed to the ease of unwinding at GATC sites in oriC.

Adenine↗
PubMed1988

Two genes, pemK and pemI, responsible for stable maintenance of resistance plasmid R100.

Plasmid R100 was found to have two genes, designated pemK and pemI, that were responsible for its stable inheritance during cell division. They are located near the region that is essential for autonomous replication. Under conditions that inhibit replication of R100 derivatives, the plasmid containing these pem genes gave only a few segregants in viable cells and increased the number of nonviable cells in the population, suggesting that a product from the pem region stabilized the plasmid by killing plasmid-free segregants. Inactivation of one of the two translational open reading frames in the pem region caused the loss of the killing function, and thus, the open reading frame is a gene designated pemK, which encodes the killing factor. The coexistence of the pem+ plasmid with a high-copy-number plasmid carrying the other open reading frame inhibited stabilization, and thus, the second open reading frame is a gene designated pemI, which encodes the inhibitor which might control the killing function of pemK. It is likely that the two open reading frames were transcribed from a promoter. There were no significant homologies in DNA sequences between the pem gene of R100 and the genes previously shown to be responsible for the stable inheritance of the other plasmids.

Amino Acid Sequence↗
PubMed1988

Identification and characterization of the products from the traJ and traY genes of plasmid R100.

The nucleotide sequence of part of the tra region of R100 including traJ and traY was determined, and the products of several tra genes were identified. The nucleotide sequence of traJ, encoding a protein of 223 amino acids, showed poor homology with the corresponding segments of other plasmids related to R100, but the deduced amino acid sequences showed low but significant homology. The first four amino acids at the N-terminal region of the TraJ protein were not essential for positive regulation of expression of traY, the first gene of the traYZ operon. The nucleotide sequence of traY shows that this gene may use TTG as the initiation codon and that it encodes a protein of 75 amino acids. Analysis of the traY gene product, which was obtained as the fusion protein with beta-galactosidase, showed that the N-terminal region of the product has an amino acid sequence identical to that deduced from the assigned frame but lacks formylmethionine. traY of plasmid F, which encodes a larger protein than the TraY protein of R100, is thought to use ATG as an initiation codon. However, a TTG initiation codon was found in the preceding region of the previously assigned traY coding frame of F. Interestingly, when translation of traY of F was initiated from TTG, the amino acid sequence homologous to the TraY protein of R100 appeared in tandem in the TraY protein of F. This may suggest that traY of F has undergone duplication of a gene like the traY gene of R100.

Amino Acid Sequence↗
PubMed1988

Differential scanning calorimetry of antitumor antibiotics-plasmid DNA interaction.

Differential scanning calorimetry (DSC) can detect stepwise melting of plasmid DNA along the molecular chain with high resolution. This method was applied to study interaction of some antitumor antibiotics with the plasmid pJL3-TB5 DNA (5277 base-pairs in length). Analysis of DSC curves of the plasmid DNA in the presence of, for example, adriamycin, an antitumor antibiotics of anthracycline group, together with theoretical analysis of the DNA melting curves obtained by calculation from the entire base sequence, led to the conclusion that adriamycin bound preferentially to the four particular regions with high G + C content. The DSC method would thus be useful for the study of properties of drugs which bind to DNA.

Antibiotics, Antineoplastic↗
PubMed1987

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.

Chromosomes, Bacterial↗
PubMed1987

Relationship between helix-coil transition and gene organization of ColE1 plasmid DNA. Differential scanning calorimetric and theoretical studies.

Differential scanning calorimetry (DSC) was carried out to analyze the transition of helix to coil state of DNA, using ColE1 DNA molecules digested with EcoRI. The DSC curves showed multimodal transition, consisting of nine to 11 peaks over a temperature range, depending on the ionic strength of the DNA solution. These DSC curves were essentially in good agreement with the optical melting curves of ColE1 DNA. The theoretical melting profiles of ColE1 DNA were predicted from calculations based on the helix-coil transition theory and the nucleotide sequence of the DNA. These profiles resembled the DSC curves and made it possible to assign the peaks seen in the DSC curves to the helix-coil transition of particular regions of the nucleotide sequence of ColE1. The helix-coil transition of each of the small genes gave rise to a single peak in the DSC curve, while the helix-coil transition of large genes contributed to two or more peaks in the DSC curve. This multimodal transition within a single coding region might correspond to the melting of individual segments encoding the different domains of the proteins. The helix-coil transition at the specific sites including ori, the origin of replication of ColE1, was also found to occur in a particular temperature range. DSC, a simple method, is thus useful for analyzing the multimodal helix-coil transition of DNA, and for providing information on the genetic organization of DNA.

Bacteriocin Plasmids↗