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H Ohtsubo

Publications and source records attributed to H Ohtsubo.

At least 73 records · Page 4Linked to original sources
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

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↗
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

Repressor gene finO in plasmids R100 and F: constitutive transfer of plasmid F is caused by insertion of IS3 into F finO.

Fertility factor F confers bacterial conjugation, a process which involves at least 20 tra genes. Resistance plasmids such as R100, R6-5, and R1 have homology with F in the tra region. Conjugal transfer of these plasmids is, however, repressed, while transfer of F is constitutive. Repression of R transfer is due to the existence of the two genes, called finO and finP; constitutive transfer of F is believed to be due to a lack of finO in F. In this paper, we report the identification and DNA sequence of the finO gene of R100, encoding a protein of 21,265 daltons. We show that F does actually encode finO, but the gene has been inactivated by insertion of IS3. Lederberg and Tatum (Nature [London] 158:558, 1946), who discovered sexuality in bacteria, may have had an Escherichia coli K-12 strain harboring such an finO F factor, which facilitated the generation of recombinant progeny useful for genetic analysis of bacteria and established the foundation for molecular genetics.

Amino Acid Sequence↗
PubMed1986

Intracranial tumors in the first year of life.

Among 100 childhood brain tumors treated at Kobe Children's Hospital from May 1970 to June 1985, 18 of the children presented with symptoms during the first year of life. This paper analyzes these 18 cases. Supratentorial tumors (78%) were more common than infratentorial ones, and 67% of all the tumors were located in the central neural axis. Initial symptoms were cranial enlargement (56%), vomiting (17%), cranial deformity (11%), blepharoptosis, respiratory distress, and ataxia. Histological diagnosis of the tumors was as follows: teratoma (3 cases), medulloblastoma (3), glioblastoma (2), astrocytoma (2), ependymoma (2), craniopharyngioma (1), choroid plexus papilloma (1), hamartoma (1), lipoma (1), melanotic progonoma (1), and an undetermined type, probably medulloblastoma (1). Seventeen of the patients underwent craniotomy for tumor resection (4 total, 4 subtotal and 7 partial removal, and 2 biopsies). Additional therapeutic methods used separately and in various combinations included ventriculoperitoneal shunt, subduralperitoneal shunt, ventricular drainage, radiotherapy and chemotherapy. Nine patients died (average 98 days) after surgery. Of the 9 survivors, 6 are still alive after more than 5 years. Five of the 6 are mentally retarded and 4 are physically handicapped to some degree.

Brain Neoplasms↗
PubMed1986

Characterization of the gene products produced in minicells by pSM1, a derivative of R100.

At least ten polypeptides larger than 6 kilodaltons (K) are produced in minicells from the miniplasmid pSM1 in vivo. pSM1 (5804 bp) is a small derivative of the drug resistance plasmid R100 (ca. 90 kb) and carries the R100 essential replication region as well as some non-essential functions. Cloned restriction fragments of pSM1 and plasmids with deletions within pSM1 sequences were used to assign eight of the ten observed polypeptides to specific coding regions of pSM1. Two of these polypeptides were identified as RepA1 and RepA2, proteins encoded by the essential replication region of pSM1/R100. The nucleotide sequence consisting of 885 bp outside the essential replication region is presented here. This sequence contains an open reading frame, orf4, for a protein 22.9 K in size, and one of the pSM1-encoded polypeptides was identified as the orf4 gene product. Five additional polypeptides were shown to be the products of other open reading frames mapping outside the essential replication region. Specific functions have been assigned to four of these polypeptides and tentatively to the fifth.

Amino Acid Sequence↗
PubMed1985

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

We 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.

Conjugation, Genetic↗
PubMed1984

A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1.

Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product. The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E. coli oriC consensus sequence. Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number. par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.

Autoradiography↗
PubMed1983

Distribution of insertion element IS1 in natural isolates of Escherichia coli.

Total DNAs from twelve natural isolates of Escherichia coli from animals and humans were examined by hybridization with a probe for IS1. Considerable variation in copy number was found. In the case of two strains isolated from the same individual, one strain contained no copies of IS1 and the other, much greater than 30. Evidence was also obtained for the existence of IS1-like elements (iso-IS1s) of greater than 15% sequence divergence relative to the IS1 from antibiotic resistance plasmid R100.

Animals↗
PubMed1983

Escherichia coli RNA polymerase binding sites and transcription initiation sites in the transposon Tn3.

We have identified the Escherichia coli RNA polymerase-binding sites and the transcription initiation sites in the transposon Tn3. Results from nitrocellulose filter-binding assays indicate that there are two regions within Tn3 capable of forming stable binary complexes with RNA polymerase. The two regions are a 208-bp region containing the N-terminal coding sequence of the transposase (tnpA) and repressor (tnpR) genes, and a 332-bp region containing the N-terminal coding sequence for the beta-lactamase (bla) gene. DNase I footprint analysis of the 208-bp and 332-bp fragments further defined an extended region of protection, approx. 110 bp long, located between the transposase and repressor coding regions, and an 80-bp region of protection near the N-terminal coding sequence of the beta-lactamase gene. In vitro transcription studies with fragments containing these protected regions allowed us to determine the precise transcription initiation sites for the transposase, repressor, and beta-lactamase mRNAs. The transposase and repressor mRNAs are transcribed divergently and their transcription initiation sites are separated by 80 bp. The -35 homology regions for the transposase and repressor promoters are separated by 10 bp and the -10 homology region of the transposase promoter is coincident with the recombination site (res) for the site-specific recombinase activity (resolvase) of the repressor protein, which is required for resolution of Tn3 cointegrates. We discuss the significance of this complex divergently transcribed promoter region with respect to regulation of Tn3 transposition and we propose a model for coordinated regulation of the tnpA and tnpR genes. We also compare the Tn3 tnpA-tnpR intercistronic region with that of the closely related transposon gamma delta.

Binding Sites↗
PubMed1982

Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids of Escherichia coli.

Plasmid Rsc13, a small derivative of the plasmid R1, contains a region necessary for replication as well as a complete copy (4957 bp) of the ampicillin resistance transposon, Tn3. We determined the nucleotide sequence of the replication region of Rsc13 to be 2937 bp and then compared this region (designated the 2.9-kb region) to the analogous region of pSM1, a small derivative of the plasmid R100 which has common ancestry with R1. Rsc13 and pSM1 were 96% homologous in this 2.9-kb region except for a discrete region of about 250 bp which showed only 44% homology. The sequence and distribution of nucleotide substitutions between Rsc13 and pSM1 supported a map of possible genes and sites which have previously been seen in the replication region of Rsc13 and pSM1 which showed only 44% homology. Analysis of the amino acid sequence and predicted conformation of the two RepA2 polypeptides, however, suggested that they were very similar. We proposed that the repA2 region of R1 and R100 was replaced by a substitution of a short DNA segment from another plasmid which was evolutionarily related to R1 and R100 but had more divergence. This event may have been mediated by a mechanism similar to that of gene conversion as described in eukaryotic systems.

Ampicillin↗
PubMed1982

Factors determining frequency of plasmid cointegration mediated by insertion sequence IS1.

We demonstrate that mutants with deletions at either end of the insertion sequence IS1 lose the ability to mediate cointegration of two plasmids, whereas mutants with deletions or an insertion within IS1 can mediate cointegration at a reduced frequency. These results, together with the nucleotide sequence analysis of the IS1 mutants, indicate that the two ends of IS1 (insL and insR) and two genes (insA and insB) that are encoded by IS1 are required for cointegration. Using a plasmid carrying two copies of IS1, we found that the individual IS1s mediate cointegration at different characteristic frequencies, and that each of two parts of plasmid DNA segments flanked by the two IS1s is a transposon, mediating plasmid cointegration at a unique frequency. When one IS1 was replaced with a mutant IS1, the remaining wild-type IS1 complemented the cointegration ability of the mutant IS1 as well as a resulting mutant transposon that was then flanked by a wild-type IS1 and a mutant IS1. The efficiency of this complementation reflected the characteristic ability of an individual IS1 present on the plasmid to promote cointegration. The results suggest that the IS1-encoded proteins are produced in different amounts, depending on the location of IS1 in the plasmid, and that these amounts determine the efficiency of complementation of the cointegration ability of a mutant IS1 as well as a mutant transposon. However, the location of an individual IS1 itself can also determine the frequency of cointegration in the presence of a given amount of the IS1 proteins. On the basis of the observation that the cointegration ability of a mutant IS1 is less efficiently complemented than is the ability of a mutant transposon, we also suggest that the IS1-encoded proteins can function in trans, but act preferentially on the IS1 or transposon sequence from which they are produced in promoting cointegration.

Codon↗
PubMed1982

[Effects of head movements on sound localization in pseudo-phonically reversed hearing].

Measurements of sound localization in the lateral dimension were taken with or without head movements when the observer wore the electronic pseudophone in an anechoic chamber. Tones of various frequencies as stimuli were presented from the loudspeakers located on the horizontal plane of the observer's head and on the circle, 2 m in diameter, around the observer, who pointed with his hand the position from which he thought the sound originated. The results of three subjects showed that: when the inter-aural axis of the pseudophone was 180 degrees reversed, real and apparent locations of the sound were symmetrical with respect to the median of the observer's head under the head-fixed condition, but were symmetrical with respect to the position of the observer's head as a center under the head-free condition. The implication of the results obtained was discussed.

Adult↗