Showing papers on "Replicon published in 1980"

Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti.

Abstract: A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.

Trans-complementable copy-number mutants of plasmid ColE1

Abstract: Plasmid ColE1, like many other small non-conjugative plasmids, is present in multiple copies (about 15 per chromsome equivalent) in Escherichia coli cells Because of their high copy number, the replication of such plasmids has been described as 'relaxed', even though there is good evidence that it is strictly controlled: ColE1 derivatives have characteristic but different copy numbers and ColE1 copy-number mutants have been characterised No plasmid-specified protein is essential for the replication of ColE1 and related plasmids, as extensive replication can occur in chloramphenicol-treated cells, in plasmid-free chloramphenicol-treated cells transfected with a hybrid ColE1/phage replicon and in vitro in extracts derived from plasmid-free cells Nevertheless, it is possible that a plasmid-specified protein is involved in ColE1 replication control in viable cells Here we show that deletion of a given non-essential region from ColE1-like plasmids results in a raised copy number Such plasmids are stably maintained and have their copy number returned to normal when a complementing plasmid is present in the same cell, indicating that a plasmid-specified diffusible gene product regulates the plasmid content of ColE1-containing cells Deletion of the equivalent region from the cloning vector pBR322 gives a derivative which has a raised copy number and which has also lost its origin for conjugal transfer; unlike pBR322, it cannot be mobilised

Recombinant DNA process utilizing a papilloma virus DNA as a vector

Abstract: A novel method and composition are provided for the replication and expression of exogenous genes in eukaryotic cells. A segment of a papilloma virus genome capable of extrachromosomal replication is linked to the foreign gene(s) using recombinant DNA techniques to provide a biologically functional replicon with a desired phenotypical property. The replicon is inserted into a eukaryotic cell by transformation, and the isolation of transformant provides cells for replication and expression of the DNA molecules present in the modified plasmid. The transforming region of the bovine papilloma virus provides a unique vector in that it provides both the capability of autonomous extrachromosomal replication but also the malignant transformed phenotype. Thus, genes which of themselves provide no selectable phenotypical property can be conveniently and efficiently introduced into eukaryotic cells and the transformants selected. The method is useful in that the foreign DNA is faithfully expressed and the gene products (proteins), such as pro-insulin (an insulin precursor) is synthesized.

A replication model for sister-chromatid exchange

Abstract: A model for the production of sister-chromatid exchanges is presented, based on the idea that double-strand breaks are generated at junctions between a completely duplicated replicon cluster and a partially duplicated replicon cluster. Agents that induce absolute blocks to DNA fork displacement will cause this condition to persist longer than normal, whereas agents that inhibit initiation of whole clusters will rarely cause it at all. During the blunt-end repair of the double-strand breaks, sister-chromatid exchange would be initiated when daughter strands of a duplicated cluster recombine with the parental strands of the partially replicated cluster. When the latter finishes replication, sister-chromatid exchange would be completed.

Autonomously replicating sequences in Saccharomyces cerevisiae

Abstract: A method is presented for isolating DNA segments capable of autonomous replication from Saccharomyces cerevisiae chromosomal DNA based on the differential transforming ability of autonomously replicating plasmids and nonreplicating plasmids. DNA plasmids that are capable of self-replication in yeast transform yeast spheroplasts at about 1000-fold higher frequency than nonreplicating plasmids. We have cloned from total yeast DNA a number of DNA segments that permit the pBR322 plasmid carrying the yeast LEU2 gene to replicate autonomously. These plasmid clones are characterized by their ability to transform Leu- spheroplasts to Leu+ at a high frequency and their ability to replicate autonomously. Analysis of the insert DNAs carried in some of these self-replicating plasmids divides them into two categories: those that are unique in the yeast genome and those that are repetitive.

Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance

Abstract: The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determined for the BglII, PstI, and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, we deleted nonessential regions of RK2 by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained five HaeII fragments of RK2 which total 5.4 kilobases. These fragments were derived from three regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kilobases) has the properties expected of the origin of replication. The outer four fragments, located in two separate regions of RK2, were found to provide, in trans, functions that permit the replication of the HaeII fragment carrying the origin of the replication. These results indicate that at least two plasmid-encoded genes, capable of acting in trans, and a replication origin are required for RK2 replication and maintenance.

Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication

Abstract: A DNA fragment containing the replication origin of the Escherichia coli K-12 chromosome was inserted in two orientations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA+ cells, whereas pBR322 replicates only in polA+ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function) of the E. coli origin.

Cloning of replication, incompatibility, and stability functions of R plasmid NR1.

Abstract: The region of R plasmid NR1 that is capable of mediating autonomous replication was cloned by using EcoRI, SalI, and PstI restriction endonucleases. The only EcoRI fragment capable of mediating autonomous replication in either a pol+ or a polA host was fragment B. SalI fragment E joined in native orientation with the part of SalI fragment C that overlapped with EcoRI fragment B, and also two contiguous PstI fragments of sizes 1.6 and 1.1 kilobases from EcoRI fragment B-mediated autonomous replication. When these individual SalI fragments were cloned onto plasmid pBR313 or the individual PstI fragments were cloned onto plasmid pBR322, none of these single fragments could rescue the replication of the ColE1-like vectors in a polA host, even in the presence of a compatible "helper" plasmid derived from a copy mutant of NR1. In contrast to the results reported for closely related R plasmid R6, EcoRI fragment A of NR1 could not rescue the replication of ColE1 derivative RSF2124 in a polA(Am) mutant or in a polA(Ts) mutant at the restrictive temperature. Although capable of autonomous replication, EcoRI fragment B of NR1 (or smaller replicator fragments cloned from it by using other restriction enzymes) was not stably inherited in the absence of selection for the recombinant plasmid. When EcoRI fragment B was ligated to EcoRI fragment A of NR1, the recombinant plasmid was stable. Thus, EcoRI fragment A contained a stability (stb) function. The stb function did not act in trans since EcoRI fragment B was not stably inherited when a ColE1 derivative (RSF2124) ligated to EcoRI fragment A was present in the same cell. A cointegrate plasmid consisting of EcoRI fragment B of NR1 ligated to RSF2124 was also not stably inherited, whereas only EcoRI fragment B was unstable when both RSF2124 and EcoRI fragment B coexisted as autonomous plasmids in the same cell. The incompatibility gene of NR1 was shown to be located within the region of overlap between SalI fragment E and the PstI 1.1-kilobase fragment. A copy mutant of NR1 (called pRR12) was found to have greatly reduced incompatibility with NR1; this Inc- phenotype is cis dominant.

Control of Plasmid R1 Replication: Functions Involved in Replication, Copy Number Control, Incompatibility, and Switch-off of Replication

Abstract: A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42°C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42°C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 × 106), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.

Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13

Abstract: The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome. Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322. Replication of these hybrid plasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus. Thus M13-infected polA- Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin. Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA. Rare transducing phages carrying single-stranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance. Plasmids containing a 270-nucleotide fragment from the gene II-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions. A central Hae III fragment, Hae III-G, containing the nucleotide sequence coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M13-infected polA- cells but not for high frequency transformation. Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA.

Initiation of DNA replication in a ColE1-type plasmid: isolation of mutations in the ori region.

Abstract: We have constructed a plaque-forming hybrid phage, lambda SN4, which behaves as a composite replicon of the lambda phage and a mini-ColE1 plasmid. From the hybrid phage, plaque-type mutants altered in the ability to replicate as a ColE1 replicon were isolated. These mutations were designated as cer, signifying ColE1 replication defective. One of such mutants, lambda SN4cer6, was studied further. The mutant DNA was unable to replicate in vivo if expression and function of its lambda replicon were inhibited. The defect could not be complemented in trans. DNA sequence determination of the mutant phage revealed a single base pair (bp) alteration, C-G to T-A, at 160 bp upstream from the ori site of its ColE1 replicon. From lambda SN4cer6, revertants were obtained that had regained function of the ColE1 replicon, and they could be classified into two groups that showed a full and a partial recovery in the rates of ColE1-driven DNA synthesis. DNA sequence determination of revertant DNA indicated that the former group contained true revertants, T-A to C-G, at the cer6 site, whereas one of the partial revertants was found to sustain a secondary-site mutation, G-C to A-T at 187 bp upstream of the ori site. It was possible to construct a hairpin structure that starts by hydrogen bonding of bases at the site -160 and -187.

F plasmid provides a function that promotes rec A-independent site-specific fusions of pSC101 replicon

Abstract: Recently, it has been reported that the tetracycline (Tc) resistance plasmid pSC101 (ref. 1) can undergo integrative recombination (that is, fusion) with a plasmid or bacteriophage genome that lacks extensive DNA sequence homology with pSC101. Such fusion of pSC101 with a second replicon can occur in the absence of the bacterial rec A gene function, and seems to involve DNA sequences on pSC101 that closely resemble the inverted repeat termini of the transposable genetic element, Tn3 (refs 2, 3). In both of the observed instances of replicon fusion involving pSC101, the fertility plasmid F was present in the bacterial cell; in one case an autonomously replicating F plasmid was used to support the infection of the male-specific bacteriophage fl (ref. 2), whereas in the other F was integrated into the chromosome of an Hfr bacterial strain being used to study mobilization of a non-conjugative plasmid4. However, in many studies with the pSC101 plasmid carried out in the absence of F in our laboratory and elsewhere, pSC101 has not been observed to undergo fusion to a second concurrently present replicon. These observations suggested to us that the F plasmid might carry a function that enables pSC101 to undergo replicon fusion. We report here results indicating that the F plasmid does, in fact, provide a trans-acting function that promotes rec A-independent site-specific recombination by pSC101, and that the function carried by F is located in the segment of the plasmid that includes the tra genes and the γδ sequence.

The Genetic Basis of the Spread of $\beta$ -Lactamase Synthesis Among Plasmid-Carrying Bacteria

Abstract: Despite the fact that $\beta$ -lactamases from a range of bacterial species - Gram-positive and Gram-negative - show evolutionary relatedness, there is no set pattern to the genetic organization that underlies their synthesis and its regulation. Thus, for example, the enzymes of many Gram-positive species are extracellular and inducible, whereas their counterparts in Gram-negative bacteria are often produced constitutively into the periplasmic space of the cells concerned. Nor is the location of the $\beta$ -lactamase genes always the same: in Escherichia coli and Staphylococcus aureus, for example, these are commonly plasmid-borne, whereas with other species the genes are chromosomal. Furthermore, the location may not be fixed: some strains of a species may, for example, have their $\beta$ -lactamase genes on a plasmid, whereas others of the same species may carry the same genes as part of their chromosome. In many cases the highly flexible genetic arrangement that underlies $\beta$ -lactamase synthesis derives from two main features: first, where plasmids are involved, their ability to be transferred to related species, and the fact that they can often replicate in their new hosts, ensure that the genes specifying a given type of $\beta$ -lactamase may move from species to species. Thus one finds enzymes of the same type in many distinct strains and species. The second source of flexibility is that the gene concerned is often part of a transposon: a genetic element incapable of independent replication, but which can move from one bacterial replicon to another by a mechanism independent of normal generalized recombination. Thus, with many $\beta$ -lactamases - as also with enzymes that inactivate other antibiotics - their genes may move from replicon to replicon within a given bacterial cell, and from cell to cell within a bacterial population. This, then, is an arrangement of much evolutionary potential: something which is operated upon by selection pressure to give rise to the resistant bacterial populations which cause so much trouble in our hospitals. In this context, moreover, one can even think of a third level of organization where plasmid-carrying bacteria move from one host to another by a process of cross-infection. Even though it is clear that some $\beta$ -lactamase genes can spread rapidly in susceptible bacterial populations, there also exist mechanisms that limit the extent to which the spread of both plasmids and transposons occurs. For example, some strains are poor recipients for certain types of plasmid, and, at a lower level of organization, some plasmids are relatively immune to the transposition of $\beta$ -lactamase transposons. Overall, therefore, as common with evolutionary systems, the performance of the system as it affects $\beta$ -lactam resistance is dependent on a balance of positive and negative influences: transfer of plasmids and transposons, on the one hand, and immunity to such transfers on the other.

In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174

Abstract: The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E. coli.

Integration of specialized transducing bacteriophage lambda cI857 St68 h80 dgnd his by an unusual pathway promotes formation of deletions and generates a new translocatable element.

Abstract: Molecular and genetic studies have revealed that several illegitimate recombinational events are associated with integration of the specialized transducing bacteriophage lambda cI57 St68 h80 dgnd his into either the Escherichia coli chromosome or into a plasmid. Most Gnd+ His+ transductants did not carry the prophage at att phi-80, and 10% were not immune to lambda, i.e., "nonlysogenic." Integration of the phage was independent of the phage Int and Red gene products and of the host's general recombination (Rec) system. In further studies, bacterial strains were selected which carried the phage integrated into an R-factor, pSC50. Restriction endonuclease analysis of plasmid deoxyribonucleic acid (DNA) purified from these strains showed that formation of the hybrid plasmids resulted from recombination between a single region of pSC50 and one of several sites within the lambda-phi 80 portion of the phage. Furthermore the his-gnd region of the phage, present in the chromosome of one nonlysogenic transductant, was shown to be able to translocate to pSC50. Concomitant deletion of phage DNA sequences or pSC50 DNA was frequently observed in conjunction with these integration or translocation events. In supplemental studies, a 22- to 24-megadalton segment of the his-gnd region of the chromosome of a prototrophic recA E. coli strain was shown to translocate to pSC50. One terminus of this translocatable segment was near gnd and was the same as a terminus of the his-gnd segment of the phage which translocated from the chromosome of the nonlysogenic transductant. These data suggest that integration of lambda cI857 St 68 h80 dgnd his may be directed by a recombinationally active sequence on another replicon and that the resulting cointegrate structure is subject to the formation of deletions which extend from the recombinationally active sequence. Translocation of the his-gnd portion of the phage probably requires prior replicon fusion, whereas the his-gnd region of the normal E. coli chromosome may comprise a discrete, transposable element.

Chromosomal rearrangements induced by temperate bacteriophage D108.

Abstract: As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriophage D108 induces chromosomal rearrangements (replicon fusion and transposition of chromosomal segments) in its host genome.

Effects of araC and aphidicolin on DNA chain elongation rate in HeLa S3 cells.

Abstract: The effects of araC and aphidicolin on DNA chain elongation rate were tested. The rate was markedly reduced at low concentrations. Total DNA synthesis was more inhibited, indicating a role of DNA polymerasea in replicon initiation.

Cloning of threonine operon genes in Escherichia coli cells

Abstract: A set of hybrid plasmids carrying Escherichia coli threonine genes was obtained and cloned. The plasmid pBR322 was used as a vehicle. The genetic and restriction analyses showed that genes thrA and thrB were placed between SalGI and EcoRI sites on the 2.6 megadaltons DNA region. The transcription of threonine operon genes inserted in the hybrid plasmids is under the control of its own promoter. The copy number of hybrid plasmids was reverse proportional to their molecular weight and did not depend on the replicon number. Amplification of genes of threonine operon by hybrid plasmids led to 20-25-fold increase of homoserine dehydrogenase activity, encoded by thrA gene. The expression of this gene, incorporated in hybrid plasmids, was repressed by the addition of threonine and isoleucine in the culture medium.

[66] Mapping the origin and terminus of replication of simian virus 40 DNA by pulse labeling

Abstract: Publisher Summary This chapter discusses the techniques for mapping the origin and terminus of replication of simian virus 40 (SV40) DNA by pulse labeling The chromosome of simian virus 40 represents a unique replicon in the infected permissive cells The replication process of the viral chromosome involves initiation, elongation, and termination One approach to localization of functional sites in the viral genome that are involved in the DNA replication steps is through the use of a pulse labeling technique A brief pulse of 3 H-thymidine administered to cells that are actively synthesizing SV40 DNA, results in incorporation of the radiolabel at the growing points of the viral DNA Molecules that are completed during the pulse contain highest labeled radioactivity at or near the terminus, whereas the specific label is lowest at or near the origin of replication 3 H-Thymidine pulse-labeled form I DNA is mixed with uniformly labeled [ 32 P]DNA and analyzed by restriction enzyme digestion From the distribution of radiolabel ( 3 H/ 32 P ratio) in fragments of known position in the genome, the origin, the terminus, and the direction of replication of the viral DNA can be deduced

Similar size of the replicons in Chironomus polytene chromosomes at two developmental stages.

Abstract: The size of the DNA replication units in salivary gland polytene chromosomes of Chironomus arvac at 2 developmental stages (red-head and late 4th instar) was investigated. The 2 developmental stages differ with respect to intensity of DNA replication with much lower values at the red-head stage. Also the level of polytenization is lower at the red-head stage. The size of the replication units was measured by determining the size of double-stranded DNA fragments released from active replication units during the cell lysis. At both stages it is possible to release from the polytene chromosome a double-stranded DNA fragment of similar size. This indicates that there is no or very little difference in size of active replication units in the same tissue at the 2 developmental stages.