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PubMed1983

Copy number mutations (Cop-) of the plasmid containing the replication origin (oriC) of the Escherichia coli chromosome: lethal effect of the cop region cloned onto a high-copy-number vector on host cells.

High-copy-number mutants were isolated from an oriC plasmid. They carried insertion mutations within a region (about 470 base pairs) near the uncB gene. When a segment containing this region was cloned onto a high-copy-number plasmid, such a plasmid could be maintained as an intact form only when it was present in a lower copy number.

Chromosomes, Bacterial↗
PubMed1983

Recognition sites for a membrane-derived DNA binding protein preparation in the E. coli replication origin.

The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is located in the minimum oriC (35-270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3'OH-5'P in the direction of the E. coli genetic map is recognized, at the second site the 5'P-3'OH strand.

Bacterial Proteins↗
PubMed1978

Map location of the Escherichia coli origin of replication.

Working with restriction fragments obtained directly from the Escherichia coli K12 chromosome, the EcoRI-HindIII restriction map of the section of the chromosome containing the replication origin has been extended by 14 kilobase pairs (kb) to cover 56 kb. Within this newly mapped portion, the ilv and rrnC cistrons have been identified by (1) hybridization of individual restriction fragmanents to the ilv-transducing phage lambdadilv5 and (2) a comparison of the restriction map of this region with the EcoRI map of lambda dilv5 and the Hind III map of the plasmid pJC110, a ColEl-ilv hybrid. The replication origin is located approximately 30 kb from the ilvE gene and 20 kb from the rrnC 16S rRNA cistron. This places the origin near 82.7 min on the genetic map, close to uncA.

Chromosome Mapping↗
PubMed1978

Mapping by interspecies transformation experiments of several ribosomal protein genes near the replication origin of Bacillus subtilis chromosome.

Bacillus subtilis 168 was transformed with DNAs from B. amyloliquefaciens K or B. licheniformis IAM 11054. These two species show a considerable difference in ribosomal proteins from B. subtilis. Analyses of the transformants indicated that the genes for 16 proteins, S3, S5, S8, S12, S17, S19, BL1, BL5, BL6, BL8, BL14, BL16, BL17, BL22, BL23 and BL25 are located in the cysA-str-spc region on B. subtilis chromosome. The genes for 10 proteins, S4, S6, S13, S16, S20, BL15, BL18, BL20, BL24 and BL28 could not be found in this region in the present experiments.

Bacillus↗
PubMed1986

The nucleotide sequence of the mitochondrial DNA genome of an abundant petite mutant of Saccharomyces cerevisiae carrying the ori1 replication origin.

We have determined the 903 bp nucleotide sequence of the mitochondrial DNA genome of a Saccharomyces cerevisiae petite mutant BB5. This petite, containing the 265 nucleotide ori1 region, is representative of a class of petites arising at exceptionally high frequency within the population of spontaneous petites derived from a particular mit- strain Mb12. The DNA sequences of both the ori1 region and the flanking intergenic regions have been compared to those of the corresponding regions of mtDNA in a previously reported petite strain, a1/1R/1 of Bernardi's laboratory, that has a similar (880 bp) repeat unit. The BB5 petite genome carries a canonical ori1 sequence that is identical in both petite mtDNAs, but the flanking intergenic sequences show significant differences between the two petite strains. The divergence is considered to arise from differences in the sequences flanking ori1 in the respective parent strains.

Base Sequence↗
PubMed1985

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. IV. Transcription of the oriC region and expression of DNA gyrase genes and other open reading frames.

We have determined nucleotide sequence of some 10,000 base pairs (bp) in the oriC region of the Bacillus subtilis chromosome. Initiation sites of transcription from this region were determined in vivo by the S1-mapping method. Five major initiation sites were found in the leader sequences of five open reading frames (ORF) deduced from the nucleotide sequence. The sixth site is located inside the ORF323("recF"). Putative promoters were found for each transcript. Function of these promoters was demonstrated in Escherichia coli by the Maxi-cell method using appropriate fragments cloned in pBR vectors. Based on these results, genes in 10,000 bp oriC region are divided into 4 transcriptional units. GyrB composes one unit with two other ORFs, while gyrA constitutes a single unit by itself. The promoters for ORF446("dnaA") and ORF378("dnaN") are located within the putative signal sequences for oriC. Transcription from these promoters is dependent on a dna-initiation gene, dnaB.

Bacillus subtilis↗
PubMed1991

New pUC-derived cloning vectors with different selectable markers and DNA replication origins.

Four new Escherichia coli cloning vectors are described, pUC6S, pUC21, pUK21 and pOK12. These vectors contain a polylinker or multiple cloning site (MCS) with the recognition sequences for 28 restriction enzymes. Plasmids pUC21, pUK21, and pOK12 contain the MCS in the N-terminal end of the lacZ alpha fragment allowing blue/white screening for inserts. To potentially increase the stability of some inserts that may encode toxic proteins, the strength of the lacZ promoter present on these vectors has been reduced. Plasmids pUC6S and pUC21 carry the bla gene encoding ampicillin resistance, while pUK21 and pOK12 contain the gene encoding kanamycin resistance. Plasmid pOK12 carries the replicon from P15A, resulting in a lower copy number pUC-type vector. Plasmid pUC6S carries the ori and bla gene present on all pUC vectors, but does not contain any lac sequences. Plasmids pUC21 and pUK21 contain the M13 intergenic region allowing for the production of plasmid single-stranded DNA. To improve the yield of ss plasmid DNA, two plasmid cis-acting factors that affect yield were also examined: the effect of plasmid-derived transcription across the M13 ori, and the effect of delecting the M13 minus-strand ori from the plasmid.

Base Sequence↗
PubMed1989

Mutational analysis of the primer RNA template region in the replication origin (oric) of bacteriophage G4: priming signal recognition by Escherichia coli primase.

The primase-dependent phage G4 origin of complementary DNA strand synthesis (G4oric) contains three stable stem-loops (I, II, and III) upstream from the initiation point of primer RNA (pRNA). Site-directed mutagenesis was used to introduce alterations into the nucleotide (nt) sequence of the G4oric pRNA template region. Mutations in stem-loop I, that changed the length of the stem and the sequence of the loop, slightly depressed, but did not abolish, G4oric activity. However, functional G4oric activity was destroyed when the sequence containing the starting position of pRNA synthesis was deleted, or when insertions were introduced between the pRNA starting position (5'-CTG-3') and stem-loop I. Reintroducing a CTG as part of a PstI linker close to stem-loop I, however, resulted in recovery of G4oric functional activity. These results suggest that the specific nt sequence, containing 5'-CTG-3', between nt 3994 and 4007, and also the distance between the starting position of pRNA synthesis and stem-loop I, are essential structural features for G4oric function.

Base Sequence↗
PubMed1980

Site-specific integration of an F' lac pro factor in the region of the replication origin (oriC) of E. coli.

An episome, F128, which carries approximately 8 x 10(4) base pairs of chromosomal DNA homologous to the lac pro region of the E. coli chromosome, has been found to integrate into the oriC region of the chromosome in a site specific reaction. While the event appears to be recA-dependent, no homology between the episome and this region of the chromosome was detected. The Hfr strains formed result from the integration of intact F128 molecules. The structure of the Hfr strains generated has been determined and their transfer properties analyzed.

Base Sequence↗
PubMed1987

Incorporation of 5-bromodeoxycytidine in the adenovirus 2 replication origin interferes with nuclear factor 1 binding.

We have studied the binding of nuclear factor 1 (NFI), a human sequence-specific DNA-binding protein, to a DNA fragment substituted in vitro with 5-bromodeoxycytidine (5-BrdC). Even at low substitution grades binding of NFI to its recognition sequence was considerably lower than with the unsubstituted control fragment. We developed a procedure to cleave substituted DNA specifically at a BrdC residue and searched for contacts between NFI and 5-BrdC residues by an interference assay. Surprisingly, no specific contacts were found in or near the recognition sequence. It appeared instead that interference was inversely related to the distance of a 5-BrdC residue from the NFI binding site. Models to explain these results, including a possible sliding mechanism, are discussed.

Adenoviridae↗
PubMed1981

The nucleotide sequence of the replication origin of plasmid NTP1.

The sequence of the DNA of the origin region of NTP1 has been obtained. Analysis of the sequence indicates that: (1) there is great sequence homology in the DNA upstream from the origin in NTP1, ColE1, CLODF13, PBR345 AND PBR322; (2) only seven base pairs of NTP1 are identical with the sequence downstream from the origin in ColE1, although some homology exists for 140 bases downstream; (3) two ten base pair direct repeats are present in NTP1 which are also conserved in all four plasmids named above; (4) probably no polypeptide greater than fifteen amino acids in length is encoded by the NTP1 origin region, since no single open reading frame is conserved in all five plasmids.

Base Sequence↗
PubMed2000

Strand symmetry around the beta-globin origin of replication in primates.

Certain mutations are known to occur with differing frequencies on the leading and lagging strands of DNA. The extent to which these mutational biases affect the sequences of higher eukaryotes has been difficult to ascertain because the positions of most replication origins are not known, making it impossible to distinguish between the leading and lagging strands. To resolve whether strand biases influence the evolution of primate sequences, we compared the substitution patterns in noncoding regions adjacent to an origin of replication identified within the beta-globin complex. Although there was limited asymmetry around the beta-globin origin of replication, patterns of substitutions do not support the existence of a mutational bias between the leading and lagging strands of chromosomal DNA replication in primates.

Animals↗
PubMed1994

Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication.

The mechanism by which a replicator (origin of replication) becomes denatured during the initiation of replication is not understood for any prokaryotic or eukaryotic system. To address this question, we chemically probed the molecular contacts on the SV40 origin of replication (ori) that are used by the SV40 large T-antigen and a single-stranded DNA-binding protein (SSB) during ori denaturation. Prior to the actual denaturation step, the T-antigen double hexamer bound ori utilizing sugar-phosphate contacts that were located on opposite strands in each flanking domain of ori. Each set of flanking phosphate contacts were also located on approximately opposite faces of the ori duplex. While the phosphate contacts had a 2-fold symmetry with respect to the ori center, T-antigen contacts with nucleotide bases were polar with critical interactions detected in only one of the two flanking domains. During origin denaturation catalyzed by T-antigen and a SSB, numerous new contacts to flanking phosphates were observed on the strand not initially bound by T-antigen, suggesting movement of each T-antigen hexamer outward from ori. These data suggest that T-antigen initially binds ori in a manner that facilitates transfer of each T-antigen hexamer to opposite strands during the initiation of SV40 replication.

Animals↗
PubMed1997

An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeast Yarrowia lipolytica.

Two DNA fragments displaying ARS activity on plasmids in the yeast Yarrowia lipolytica have previously been cloned and shown to harbor centromeric sequences (P. Fournier, A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J.-W. Xuan, A. Peito, A.-M. Ribet, C. Feynerol, F. He, and C. Gaillardin, Proc. Natl. Acad. Sci. USA 90:4912-4916, 1993; and P. Fournier, L. Guyaneux, M. Chasles, and C. Gaillardin, Yeast 7:25-36, 1991). We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not.

Base Sequence↗