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S Hiraga

Publications and source records attributed to S Hiraga.

At least 199 records · Page 11Linked to original sources
PubMed1983

Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome.

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions. The ends were then labeled selectively with T4 polynucleotide kinase and [gamma-32P]ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.

Base Sequence↗
PubMed1983

A novel type of E. coli mutants with increased chromosomal copy number.

We have isolated E. coli mutants which can grow at 30 degrees C but not at 42 degrees C and are able to harbor the oriC plasmid (minichromosome) at a higher copy number than the parental wild-type strain at the permissive temperature. The mutants were found to contain higher amounts of chromosomal DNA per mg protein than the wild-type, whether or not they harbor the plasmid. Experimental results suggest that the higher amount of chromosomal DNA is due to a higher copy number of chromosomes and not to a larger amount of DNA per chromosome. These properties in each of the mutants are caused by a single mutation at the rpoB or rpoC gene that code for the beta or beta' subunit of RNA polymerase, respectively. The mutations are thought to affect the regulation of replication of oriC-bearing replicons, that is, the E. coli chromosome and oriC plasmids, but not the miniF plasmid.

Bacterial Proteins↗
PubMed1983

Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition.

Plasmids that replicate using the replication origin (oriC) of the E. coli chromosome are not stably inherited through cell division, but can be stabilized by joining with a particular segment of F plasmid that presumably provides the partition function. The segment necessary for stabilization has been located within a 3.0 kb segment outside of the region essential for autonomous replication of the F plasmid. This segment contains three functionally distinct regions: two of them (designated sopA and sopB) specify gene products that act in trans, whereas the third region (sopC) acts in cis. All three functions seem to be essential for normal partition of the plasmid into daughter cells during cell division. The cis-acting region also specifies plasmid incompatibility.

Bacterial Proteins↗
PubMed1983

Mini-F plasmid genes that couple host cell division to plasmid proliferation.

A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid. An oriC plasmid carrying both a mini-F segment necessary for partition [coordinates 46.4-49.4 kilobase pairs (kb) on the F map] and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the parental mini-F plasmid. When replication of a plasmid carrying ccd is prevented and the plasmid copy number decreases, to as few as one per cell, host cell division is inhibited, but not increase of turbidity or chromosome replication. Appearance of plasmid-free segregants is therefore effectively prevented under such conditions. Experimental results suggest that reduction of the copy number of plasmids carrying the ccd region causes an inhibition of cell division and that the ccd region can be dissected into two functional regions; one (ccdB) inhibits cell division and the other (ccdA) releases the inhibition. The interplay of the ccdA and ccdB genes promotes stable plasmid maintenance by coupling host cell division to plasmid proliferation.

Base Sequence↗
PubMed1983

[Nuclear DNA].

Explore the source record for details and available documents.

Animals↗
PubMed1982

In vitro replication of recombinant plasmids carrying chromosomal segments of Xenopus laevis.

Recombinant plasmids carrying a segment of Xenopus laevis chromosomal DNA were constructed with plasmid pBR322 as the vector. A recombinant plasmid pXY65 carrying a 3.2-kilobase BamHI segment of the chromosome of X. laevis has been found to contain a repetitive sequence dispersed throughout the X. laevis chromosomes. This plasmid initiated replication in vitro when the supercoiled circular molecules were incubated in a replication system. The other recombinant plasmids tested and the pBR322 vector were not replicated. Electron microscopic analysis of the replicative intermediates showed that the replication was initiated at a specific site in the 3.2-kilobase BamHI segment of pXY65 and that the replication usually proceeded bidirectionally. Analysis of the reaction products by centrifugation in alkaline sucrose gradients indicated that short pieces were synthesized in the in vitro replication system. DNA synthesis was inhibited in vitro by the addition of aphidicolin and by omission of dNTPs. These results indicate that the X. laevis segment cloned in pXY65 contains a site capable of initiating replication in vitro.

Animals↗
PubMed1980

Copy-number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication.

A composite plasmid (pXX11) was constructed by joining of an oriC plasmid (pMCR115) carrying the replication origin (oriC) of the Escherichia coli chromosome and a mini-F plasmid (pSC138) carrying the ampicillin-resistance gene (bla). Plasmid pXX11 can replicate, by using oriC, in Hfr cells and mafA mutant cells that cannot support replication of an F plasmid. This plasmid is stably maintained in these host cells during cell growth even under nonselective conditions by use of the partition mechanism of the mini-F genome. In contrast to other oriC plasmids reported previously, pXX11 has no detectable effect on host cell growth. Higher copy-number (Cop-) mutants of pXX11 were isolated, and some of them were found to carry an insertion or deletion within a region derived from the E. coli chromosome. This region, designated cop (copy number), covers about 0.7 kilobase pair and is located approximately 3 kilobase pairs away from the oriC region at the side opposite the asn gene. Evidence suggests that the normal cop region locted on the oriC plasmid acts to reduce the copy number of the plasmid. Plasmid pXX11 complements the uncB402 mutation located on the host chromosome, but some of the Cop- plasmids do not, suggesting that the cop region is vey closely linked to uncB.

DNA Replication↗
PubMed1979

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.

Chromosome Mapping↗