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H Ohtsubo

Publications and source records attributed to H Ohtsubo.

87 records · Page 5Linked to original sources
PubMed1981

Role of RNA transcripts in replication incompatibility and copy number control in antibiotic resistance plasmid derivatives.

The genes required for autonomous replication and incompatibility in the antibiotic resistance plasmids R100 and R1 have been located within a 2.5-kilobase region of the 90-kilobase genome, within which the incompatibility gene occupies a 1.3-kilobase region excluding the replication origin. We now report that three RNA species are synthesized in vitro from the 2.5-kilobase region, which R100 and R1 have in common. One, a long RNA molecule which is transcribed in the direction of DNA replication, probably acts as a messenger or a protein required for plasmid replication. The second RNA species, only 91 nucleotides long, is transcribed in the opposite direction, from a region of the DNA entirely contained within the first and known to specify incompatibility and copy control functions. The third RNA species, 150 bases long, is transcribed from a region including the replication origin; it may be a primer of DNA synthesis or, in conjunction with the second of the three RNA species, an influence in the control of replication.

Chromosome Mapping↗
PubMed1981

Distribution of the insertion sequence IS1 in gram-negative bacteria.

Translocation of DNA segments is a recombinational event seen in both eukaryotic and prokaryotic chromosomes, and it is thought to be involved in controlling gene expression and in the evolution of chromosomes. In bacteria, insertion (IS) and transposable (Tn) elements not only translocate their own DNA, but also promote the rearrangement of both bacterial chromosomes and the plasmic genomes carrying them. The insertion element IS1 is one such element which is 768 base pairs long. IS1 is involved in the generation of deletion mutations and in the fusion of two different plasmid genomes. It can also promote the translocation of DNA segments flanked by two copies of IS1 to give rise to transposable elements responsible for antibiotic resistance and enterotoxin production. We report here the distribution of the IS1 sequence in various bacterial DNAs, particularly in the family Enterobacteriaceae. Comparison of the results with the phylogenetic relationship of these bacteria suggests that IS1 was transferred from one bacterium to another after their divergence and in some bacteria the copy number of IS1 increased by translocation. The increase in the number of copies of IS1 in bacteria may increase the probability of the genetic rearrangement responsible for the generation of resistance and enterotoxin plasmids, the existence of which is a serious problem in medical microbiology.

Acinetobacter↗
PubMed1980

Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis.

The nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acis; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA2 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA2 either encodes a gene product or structure information that is responsible for incompatibility.

Base Sequence↗
PubMed1980

Plasmids containing insertion elements are potential transposons.

We studied in vivo recombination between the plasmid pHS1, a temperature-sensitive replication mutant carrying tetracycline resistance, and pSM1, a small plasmid carrying one copy of the insertion element IS1. Recombinant plasmids were found by selection for tetracycline resistance at 42 degrees C. Their formation was independent of recA function. Analysis of the physical structures of various recombinant DNA molecules with electron microscopy and restriction endonucleases revealed that pSMI was integrated at its IS1 into numerous sites on pHS1, giving rise to a duplication of IS1 in the same orientation at both junctions. Nucleotide sequence analysis of recombinant plasmids and their parental plasmid DNA revealed that nine nucleotides at a target site were duplicated at the junction of each IS1. This phenomenon implies that plasmids containing a translocatable DNA element can be potential transposons.

Base Sequence↗
PubMed1980

Insertion element IS102 resides in plasmid pSC101.

In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.

Base Sequence↗
PubMed1979

The nucleotide sequence of the region surrounding the replication origin of an R100 resistance factor derivative.

The replication origin of a group of small plasmids derived from R100 was previously determined by electron microscopy (Ohtsubo et al., 1977). This region was subjected to extensive restriction enzyme analysis and the nucleotide sequence of the region containing the replication origin was determined using the Maxam and Gilbert sequencing technique. Various characteristics of this sequence, including a very interesting secondary structure are described and discussed.

Base Sequence↗
PubMed1979

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.

Base Sequence↗
PubMed1978

Interaction of bleomycin with DNA.

The sequence of oligonucleotides produced by the action of bleomycin and ferrous ion on double- and single-stranded DNA has been determined. In the presence of ferrous ion, bleomycin promotes cleavage at G-T and G-C sequences, while high concentrations of ferrous ion alone result in strand scission that is not base specific. In the presence of bleomycin and ferrous ion, pyrimidine bases located to the 3' side of guanosine are released preferentially and a low molecular weight product that forms a chromophore with thiobarbituric acid is produced from the deoxyribose moiety. Oligonucleotides produced by the action of bleomycin differ slightly in electrophoretic mobility from those produced by chemical or enzymatic cleavage. A model is proposed to explain the interactions of bleomycin with DNA.

Base Sequence↗
PubMed1978

Nucleotide sequence of an insertion element, IS1.

PSM2, PSM1, and PSM15 are small plasmids derived from R100 by spontaneous deletions at either end of the insertion sequence IS1. These plasmids were used to identify regions neighboring IS1 as well as the IS1 DNA itself, by cleavage with EcoR1, HindIII, Hae III, Hpa II, Hha I, Hinf, and AIu I. The nucleotide sequencing results demonstrate that IS1 contains 768 bases. About 30 bases at the ends of IS1 were found to be repeated in an inverted order. The deletions occurring at the ends of IS1 were found to be due to illegitimate recombination. The hypothesis that RNA polymerase could play an important role in such recombination phenomena is discussed based on the nucleotide sequences surrounding the recombinational hot spots.

Base Sequence↗
PubMed1976

Isolation of inverted repeat sequences, including IS1, IS2, and IS3, in Escherichia coli plasmids.

A method is described for isolation of inverted repeat DNA sequences that occur in E. coli plasmids. The procedures of the isolation involved: (a) denaturation of intact plasmid DNA, (b) a rapid, 30 sec, renaturation of inverted-repeat sequences in the genome, (c) digestion of the single-stranded portion by S1 nuclease to recover duplex DNA, and (d) detection and purification of the duplexes using 1.4% agarose gel electrophoresis. If a plasmid DNA carried inverted repeats of either one type or two different types of special DNA sequences, these procedures enabled us to observe either one or two characteristic DNA bands, respectively, in the agarose gels. If a plasmid DNA did not carry any inverted repeats, or if the plasmid DNA only carried direct repeat sequences, no characteristic DNA bands were recovered. Cleavage of the spacer DNA between inverted repeat sequences generated no gel bands. This indicated that the inverted repeat sequences must be in the same strand. Using this method, we isolated and purified several repeated sequences, including IS1, IS2, and IS3, from derivatives of F and R plasmids.

Base Sequence↗