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Biomedical subjects

S Hiraga

Publications and source records attributed to S Hiraga.

At least 217 records · Page 12Linked to original sources
PubMed1979

Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome.

The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phase lambda i21 dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of lambda i21 dnaA-2 and pMCR501 revealed the following. The lambda i21 dnaA-2 DNA retained the delta sr I lambda 1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. The size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in lambda i21 dnaA-2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the lambda i21 dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the lambda i21 dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322.

Bacteriophage lambda↗
PubMed1979

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.

Bacterial Proteins↗
PubMed1978

Bacteriophage lambda carrying the Escherichia coli chromosomal region of the replication origin.

A transducing phage lambdaasn was isolated. The late gene region of its genome was found to have been substituted by an Escherichia coli chromosomal segment containing the genes bgIR, bgIC, glmS, uncA, and asn. Restriction endonuclease cleavage mapping and electron microscopic analysis of the lambdaasn DNA revealed that the size of the bacterial segment is approximately 1.75 X 10(7) daltons, corresponding to about 26.4 kilobases. The circular DNA of lambdaasn was digested with restriction endonuclease EcoRI, diluted, and sealed with DNA ligase. When the reaction mixture was used to transform a recipient E. coli strain, a small plasmid of about 1 X 10(7) daltons (named pMCR115) was obtained. Restriction endonuclease cleavage mapping of pMCR115 and other evidence suggested that it contained the replication origin (oriC) of the E. coli chromosome.

Chromosomes, Bacterial↗
PubMed1977

Replication of Fpoh+ plasmid in mafA mutants of Escherichia coli defective in plasmid maintenance.

A class of F' plasmids, designated Fpoh+, was previously shown to be able to replicate extra-chromosomally on Hfr strains by virtue of carrying the specific site or region poh+ (permissive on Hfr) of the E. coli chromosome (Hiraga, 1975, 1976a). These plasmids were now found to replicate on E. coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids. The derivatives of Fpoh+ that have lost the poh+ site, on the other hand, failed to replicate on mafA mutants. These mutants harboring Fpoh+ (but not Poh- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh+ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons. It is tentatively concluded that the poh+ site is required for F' plasmids to replicate on mafA mutants as well as on Hfr strains. In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh+ region contains the replication origin of the E. coli chromosome.

Conjugation, Genetic↗
PubMed1976

Novel F prime factors able to replicate in Escherichia coli Hfr strains.

A novel type of F' plasmid that can replicate extrachromosomally in cells of Hfr strains has been isolated. Genetic analysis of these plasmids indicates that all carry the dnaA-bglA region of the E. coli chromosome as well as ilv+ used as a selective marker. It is suggested that a specific site, designated poh+ (permissive on Hfr), is located in this region, and is essential for these plasmids to replicate in Hfr cells. The most interesting explanation, among others, would be that the poh+ site represents the replication origin of the bacterial chromosome.

Chromosome Mapping↗
PubMed1975

Initiation of DNA replication in Escherichia coli. II. Effect of rifampicin on the resumption of replication of F episome and chromosome upon the returning of dna mutants from a non-permissive to a permissive temperature.

When E. coli F+ cells carrying the dna-167 or dnaC2 mutation, which causes the temperature-sensitive initiation of DNA replication, are exposed to a non-permissive temperature to stop the replication of chromosome and F factor, and then transferred back to a permissive temperature with the addition of chloramphenicol, one round of the chromosomal replication occurs, but further replication is inhibited. Under these conditions, F DNA replicates coincidentally with the initiation of the chromosomal replication in both strains. When rifampicin is added to the cells upon lowering of the temperature, the chromosome can not replicate in the F+ dna-167 strain, but can do so in the F+ dnaC2 strain. F DNA can replicate in both of the mutant strains under these conditions.

Chloramphenicol↗
PubMed1975

Initiation of DNA replication in Escherichia coli. III. Genetic analysis of the dna mutant exhibiting rifampicin-sensitive resumption of replication.

Temperature-sensitive mutants defective in the initiation of DNA replication are exposed to a non-permissive temperature to complete already initiated replication, and are transferred back to a permissive temperature. DNA synthesis can resume in the presence of rifampicin or rifampicin plus chloramphenicol in strain PC2 (dnaC2), but not in strain N167 (dna-167). In the presence of chloramphenicol alone, however, DNA synthesis can resume in both strains (Hirage and Saito, 1973, 1974). The double mutants carrying the dna-167 and dnaC2 mutations show the rifampicin-sensitive resumption of DNA replication as the dna-167 mutant. The rifampicin-sensitive character (designated as Rrr-) is closely linked with the temperature sensitivity of the dna-167 mutant in P1 transduction. The gene order is dna-167-tna-phoS-uncA-ilv. The Rrr- character does not correlate with the inactivation of the altered product of the mutated dna-167 gene at various temperatures in the double mutant carrying dna-167 and dnaC2. Although dnaC2 strains show the Rrr+ phenotype, the dnaC2 strains received the ilv-dnaA region of the Ts+ revertants obtained from a dna-167 strain show the Rrr- phenotype. These results suggest that the dna-167 mutant has two mutations which are closely linked to each other, controlling the Rrr- phenotype and the temperature sensitivity, respectively.

Bacterial Proteins↗
PubMed1975

F deoxyribonucleic acid transferred to recipient cells in the presence of rifampin.

When Escherichia coli F+ cells resistant to rifampin and streptomycin are mated with F- cells sensitive to the antibiotics, a large fraction of F factor deoxyribonucleic acid (DNA), which is transferred to the recipient cells in the presence of either of the antibiotics, can be recovered as covalently closed, circular double-stranded DNA, as in the control mating in the absence of antibiotic. Similar results were obtained in the presence of chloramphenicol or chloramphenicol plus rifampin. It is suggested that the transferred single-stranded F DNA can be converted to the covalently closed, circular double-stranded form in the absence of protein synthesis, and that rifampin-sensitive transcription is not required for the conversion.

Centrifugation, Density Gradient↗
PubMed1975

F deoxyribonucleic acid superinfected into phenocopies of donor strains.

When F+ donor cells of Escherichia coli are conjugated with F-, F+, or Hfr recipients under the conditions of phenocopy mating, the male recipients are found capable of accepting the F episome as effectively as the F- recipients. The F deoxyribonucleic acid (DNA) superinfected into the male recipients is converted to the covalently closed, circular duplex form, as in the F- recipients. It is also found that the synthesis of the strand complementary to the transferred single strand and its subsequent conversion to the covalently closed, circular duplex occur effectively in male recipients as well as in female recipients. Under these mating conditions, F-ilv+ episome superinfected to F+ and Hfr cells is diluted out during growth, whereas F-ilv+ transferred into F-cells is replicated and established in almost all progeny cells. These results suggest that the incompatibility of the F episome is not due to the reduction in the rate of the conversion of transferred single-straned F DNA to covalently closed, circular duplex, but, rather, to an inhibition of further replication of the covalently closed, circular F DNA.

Centrifugation, Density Gradient↗