Structural properties of the beta origin of replication of plasmid R6K

The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.     

Aerobactin iron uptake sequences in plasmid ColV-K30 are flanked by inverted IS1-like elements and replication regions.

By using Southern blot hybridization procedures, we found that a specific sequence within a 16.3-kilobase HindIII restriction fragment of pColV-K30 was also present in at least three other pColV-K30 HindIII fragments. Restriction endonuclease mapping of these HindIII fragments indicated that two of these repeated sequences, identified as IS1-like, occur in reverse orientation adjacent to the ends of the aerobactin iron uptake region, also included in the 16.3-kilobase HindIII fragment. There are two distinct replication regions enclosing the IS1-flanked aerobactin genes. A pColV-K30 BamHI restriction endonuclease fragment, carrying one of these replication regions, showed homology with the F plasmid EcoRI fragment f5, which carries the F replication sequences.     

A comparison of the origin of replication of pSa with R6K

Summary The plasmid pOri3 is a derivative of the origin of replication of pSa. Replication is defective as a result of a truncated repA gene, the product of which is required for plasmid replication. The defective replication is complemented by the presence of the intact repA gene of pSa, or by the presence of the plasmid R6K. The basis of this complementation has been examined by comparing the nucleotide sequence of the origin of pSa with that of R6K. A 13 base pair sequence present twice in the origin of pSa has homology with a 13 base pair sequence that is present fourteen times in the origin of R6K. These sequences may be the binding sites for the initiator proteins of these two plasmids. The location of these binding sites relative to the genes for the initiator proteins suggests that an autoregulatory control mechanism for the synthesis of the initiator proteins may also play a role in the control of plasmid copy number.     

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.     

A molecular analysis of a broad-host-range plasmid isolated from Thiobacillus ferrooxidans

Abstract: A 12.4-kb plasmid, pTF-FC2, that was isolated from Thiobacillus ferrooxidans and which is capable of replication in a wide range of Gram-negative bacteria, has been sequenced. The extent of the regions involved in both replication and mobilization have been delineated. The site of initiation of replication ( oriV ) has been localized on a 185-bp fragment and the origin of transfer ( oriT ) on a 138-bp fragment. Three proteins that were essential for replication and four that were essential for mobilization have been identified. The origin of replication was clearly similar to that of the IncQ plasmids although no complementation or incompatibility between pTF-FC2 and the IncQ plasmid, R300B, was detected. There was a clear similarity in the size,location and amino acid sequence of the proteins of the pTF-FC2 mobilization region with those of the TraI region of the IncP plasmids, RP4 and R751.Two inverted repeated sequences which had 37/38-bp and 38/38-bp sequence identity with the Tn 21 transposon were identified. The C-terminal part of a transposase and the N-terminal portion of a resolvase were located between the inverted repeats. These open reading frames are most likely the remnants of a defective transposon. A protein with homology to a mercury- resistance regulator was also present within the transposon-like element although no gene encoding for mercury reductase could be indentified.     

Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase

The nucleotide sequence of the 1.30-kilobase EcoRI/BglII fragment from Vibrio harveyi carrying the majority of the luciferase beta subunit coding region (luxB gene) has been determined. The EcoRI/BglII fragment was derived from a 4.0-kilobase HindIII fragment carrying both luxA and luxB which was detected in a genomic clone bank based on the expression of bioluminescence from colonies of Escherichia coli carrying V. harveyi HindIII fragments in plasmid pBR322 (Baldwin, T. O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., and Ziegler, M. M. (1984) Biochemistry 23, 3663-3667). The entire alpha subunit coding sequence (luxA gene) and the amino-terminal 13 codons of the beta subunit sequence (luxB gene) were contained on a 1.85-kilobase EcoRI fragment, the sequence of which has been reported (Cohn, D. H., Mileham, A. J., Simon, M. I., Nealson, K. H., Rausch, S. K., Bonam, D., and Baldwin, T. O. (1985) J. Biol. Chem. 260, 6139-6146). The beta subunit coding sequence was found to terminate 972 bases past the start of the luxB coding sequence. The beta subunit had a calculated molecular weight of 36,349 and comprised a total of 324 amino acid residues; the alpha beta dimer had a molecular weight (alpha + beta) of 76,457. There were 27 base pairs separating the stop codon of the beta subunit structural gene and a 340-base open reading frame extending to (and beyond) the distal BglII site. Approximately two-thirds of the beta subunit was sequenced by protein chemical techniques. The amino acid sequence predicted from the DNA sequence, with few exceptions, confirmed the chemically determined sequence, and the measured amino acid composition was in excellent agreement with the composition implied from the DNA sequence.     

Nucleotide sequence and functional properties of DNA encoding incompatibility in the broad host-range plasmid R1162

Summary A 370 base pair (bp) fragment of R1162 DNA encoding the incompatibility determinant has been cloned and sequenced. The DNA is located between 6.1 and 6.5 on the R1162 map, near the origin of replication. The sequence contains three perfectly conserved 20 bp direct repeats, with 11 bp of this sequence repeated a fourth time. The direct repeat unit shows some homology with that of another, unrelated broad host-range plasmid, RK2. The cloned DNA has two other properties: it lowers the copy number of R1162 when cloned into this plasmid, and it is required in cis for replication of R1162 satellite plasmids.     

Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the Epstein-Barr virus R transactivator.

Immediate-early (IE) RNA 2, the less abundant of two bovine herpesvirus 4 (BHV-4) RNAs detected in Madin-Darby bovine kidney cells infected in the presence of cycloheximide, is a 1.8-kb cytoplasmic polyadenylated RNA transcribed from the 8.3-kb HindIII fragment F. The structure of IE RNA 2 has been determined by S1 nuclease and exonuclease VII mapping, primer extension analysis, and sequencing of a partial cDNA. IE RNA 2 consists of a short, approximately 60-nucleotide 5' exon spliced to a 1.8-kb 3' exon. DNA sequence analysis revealed an open reading frame encoding 551 amino acids with sequence homology to the Epstein-Barr virus (EBV) R transactivator and its homolog in herpesvirus saimiri, HVS.R.IE 2 and HVS.R show higher homology to each other than to the EBV R transactivator. The homology is highest in the approximately 320 amino-terminal amino acids. All three proteins have acidic carboxyl termini but have little amino acid sequence homology in this region. In transient expression cotransfection assays, IE 2 activated expression from the BHV-4 early promoter-regulatory region of the major DNA-binding protein homolog over 100-fold in bovine turbinate cells. IE 1 was not necessary for this transactivation and did not augment it. However, IE 2 did not transactivate EBV or herpesvirus saimiri early promoter-regulatory regions that are transactivated by the EBV R transactivator or HVS.R.     

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control.

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.     

Stably bent and low-melting DNA sequences at the origin of replication of chicken alpha-globulin gene domains]

The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site of the alpha-globin gene domain has been studied. The fragment was shown to contain the CR1-type repetitive element and two stable bent DNA sequences.     

Alu-like sequences in the regions of replication initiation in plasmids p15A and R6K]

A method of computer analysis of DNA sequences has been proposed. It is based on information similarity of compared sequences and it significantly increases the usefulness of the computer analysis. This approach has been applied to the search of interconnected areas of Alu-repeats and replication origins of p15A and R6K plasmids. An Alu-like region located in the first stem of the secondary structure of RNA-1 and E. coli RNA-polymerase binding site has been found in the p15A. On R6K replication origin, Alu-like repeats have been found in the area of tandem 22 bp repeats. This comparison also allowed to reveal hidden periodicity of the sequence of human Alu-repeat. A hypothesis that explained the data obtained has been proposed. The proposed approach may be used as a method for revealing DNA sequences that have similar genetic functions.     

Structural-functional organization of the incompatibility group P plasmid pBS221

Plasmid pBS221 was physically mapped for restriction endonucleases EcoRI, BamHI, BglII, HindIII. The regions essential for the plasmid existence and participating in replication (oriV trfA*) and mobilization (mob) were cloned. The tet determinant and oriV trfA* regions were localized on the physical map of the plasmid. A DNA sequence homologous to genes of Tn501 mer operon was detected in this plasmid. The studies on homology of plasmids RP4 (IncP alpha), R751 (IncP beta) and pBS221 plasmid suggest that the latter belongs to the IncP beta subgroup.     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Cloning and characterization of a gene encoding 22 kDa functional protein of bacteriophage MB78

Functional protein of MB78 bacteriophage having apparent molecular weight of 22 kDa is expressed from 1.7 kb HindIII G fragment. The nucleotide sequence of this fragment showed two open reading frames of 222 and 196 codons in tail-to-tail orientation separated by a 62-nucleotide intercistronic region. The ORF of 22 kDa protein is present in opposite orientation, i.e. in the complementary strand, preceded by a strong ribosomal binding site and a promoter sequence. Another ORF started from the beginning of the fragment whose promoter region and translational start site lies in the 0.45kb HincII U fragment which is located next to the HindIII G fragment, that has the sequence for DNA bending. 3'end of the fragment has high sequence homology to the EaA and EaI proteins of bacteriophage P22, a close relative of MB78 phage.     

Characterization of a phage-plasmid hybrid (phasyl) with two independent origins of replication isolated from Escherichia coli.

The phage-plasmid hybrid phasyl can replicate as a phage in the presence of a filamentous phage of Escherichia coli (M13, fl, fd). The extragenic region of phasyl shows homology with the plus and the minus origins of filamentous phages. Insertion of a Cmr fragment into the plus origin or of a Kmr fragment into the minus origin resulted in a reduced transduction frequency, while insertion into other parts of the extragenic region did not. This suggests that phagelike replication of phasyl is mediated by an origin that coincides with the two homologous elements in the extragenic region. Autonomous replication of phasyl occurs from a second origin (oriA) that is located between positions 297 and 636. This fragment mediates replication if the Arp protein is supplied in trans. Arp is the only phage-encoded protein and is essential for plasmidlike replication. No sequence homology to other known origins was found. Phasyl derivatives with either one of the two origins inactivated can be rescued via the alternative replication mode, suggesting that the two replication pathways are independent.     

Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.