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PubMed1970

Concerning the influence of integrated episomes on chromosomal replication in Escherichia coli.

Two strains of Escherichia coli K12, C600, and DG163, in which the origins and directions of DNA replication had been mapped previously, were made lysogenic for lambda and lambdai434. The origins of replication in these lysogenic strains were located with respect to the prophage location by chromosome alignment and subsequent hybridization of (3)H-labeled lambda vir DNA with newly replicated and "old" DNA. The results obtained are consistent with similar origins of replication observed in the nonlysogenic strains. These findings suggest that DNA replication in these cells is under the control of the E. coli chromosomal replicon and is independent of the presence of the lambda replicon as predicted by the Jacob, Brenner, and Cuzin model of DNA replication.

Amino Acids↗
PubMed1973

Construction of biologically functional bacterial plasmids in vitro.

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins.

Base Sequence↗
PubMed1975

Cloning, isolation, and characterization of replication regions of complex plasmid genomes.

EcoRI endonuclease-generated DNA fragments carrying replication regions of the F'lac and R6-5 plasmids have been cloned and isolated, using as a selection vehicle a nonreplicating ampicillin-resistance DNA fragment derived from a Staphylococcus aureus plasmid. Heteroduplex analysis of the constructed plasmid chimeras and the parent replicons has localized the cloned R6-5 replication region to a DNA segment between kilobase pair coordinates 1.0 and 88.0 on the R6-5 map. Physical proximity between the plasmid replication functions and the locus governing plasmid incompatibility has been shown for both parent replicons. The cloning method reported appears to be generally applicable for the identification and isolation of replication regions of a variety of complex genomes.

Centrifugation, Density Gradient↗
PubMed1978

Construction of a novel plasmid-phage hybrid: use of the hybrid to demonstrate ColE1 DNA replication in vivo in the absence of a ColE1-specified protein.

A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid ColE1. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColE1 replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColE1 DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColE1 plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that ColE1 does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.

Bacterial Proteins↗
PubMed1981

Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene.

For the eventual purpose of isolating and studying a single animal cell replicon, we have developed a methotrexate-resistant Chinese hamster ovary cell line that has amplified an early-replicating DNA sequence approximately 500 times; this sequence includes the gene coding for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). DHFR composes 30% of the cytoplasmic protein in this cell line, and DHFR mRNA represents 25% of the message translatable in vitro. After digestion of genomic DNA from resistant cells with restriction enzymes, a unique set of highly repetitive restriction fragments can be visualized on agarose gels by ethidium bromide staining. These bands are not present in digests of parental DNA. We estimate the total length of the unit repeated sequence to be 135 +/- 15 kilobase pairs. Regardless of the restriction enzyme utilized, a subset of these repetitive fragments hybridizes to radioactive DHFR cDNA. The homogeneously staining regions on mitotic chromosomes in which these amplified sequences are located are shown to be early-replicating, as are the highly repeated restriction fragments themselves. These data suggest that an early replicon can be isolated from this region, and that this entire, normally unique, genomic segment can be cloned and mapped with respect to origins of DNA synthesis and promoters for transcription, as well as other genetic features of interest.

Animals↗
PubMed1981

Resolution of cointegrates between transposons gamma delta and Tn3 defines the recombination site.

Transposition of the genetically related insertion elements gamma delta and Tn3 is thought to involve two steps. In the case of transposition from one replicon to another, the first step is fusion of the parent and target replicons with the element appearing in direct orientation at the two junctions. In a subsequent reaction, the cointegrate structure is resolved via a site-specific recombination event. I have constructed two plasmids, each carrying segments of gamma delta and Tn3, that contain the internal resolution site. The tnpR gene product encoded by either Tn3 or gamma delta mediates intramolecular recombination between these two sites. The product of this recombination is a hybrid region that contains gamma delta and Tn3 sequences fused at the point of crossover. DNA sequence analysis of such recombinants indicates that the recombination occurs within a 19-base-pair (bp) region of exact homology between gamma delta and Tn3. The site lies in the 160-bp center intercistronic region, 50 bp before the beginning of the tnpA gene. My results therefore suggest a model for the coupled regulation of the repressor (tnpR) and the transposase (tnpA) genes and site-specific recombination of transposition intermediates. The Tn3/gamma delta recombination system and bacteriophage lambda integration are compared.

Bacteriophage lambda↗
PubMed1983

Structural requirement for IS50-mediated gene transposition.

Replicative transposition is signaled by the formation of cointegrates in which donor and target replicons are joined by direct repeats of a transposable element. Elements not generating such cointegrates may move by a conservative breaking and joining process. The IS50 elements forming the terminal repeats of Tn5 [which carries the determinant for kanamycin resistance (Kanr)] contain genes and sites necessary for transposition and mediate the movement of any DNA segment they bracket. To determine if IS50 generates cointegrates, the products of transposition from pBR322::Tn5 plasmids to an F factor in recA-Escherichia coli were examined. With monomeric pBR322::Tn5 plasmids, transposition of Kanr (from Tn5) was generally not accompanied by movement of the determinant for ampicillin resistance (Ampr) (from the pBR322 vector). With dimeric pBR322::Tn5 plasmids, by contrast, half of the transpositions of kanr were accompanied by transposition of ampr. Restriction endonuclease analyses indicated that these F-Kanr Ampr chimeras contained inserts of a single copy of the pBR322 vector sequence bracketed by one Tn5 element and one IS50 element or by a pair of Tn5 elements. None of 79 chimeras tested was a true cointegrate. Because IS50 seems to move only a segment of the donor replicon it is proposed that IS50 transposition is conservative.

DNA Replication↗
PubMed1985

Mini-F plasmid-induced SOS signal in Escherichia coli is RecBC dependent.

Dispensable replicons such as F plasmid [95 kilobases (kb)] or its mini-derivatives such as mini-F (9.3 kb) or lambda mini-F efficiently induced cellular SOS genes such as sfiA (sulA) when they were damaged by UV irradiation and then introduced into a recipient bacterium. To generate an SOS signal, UV light-damaged mini-F or mini-F conditional mutants deficient in replication required that the bacterial RecBC enzyme retained some activity different from the nuclease activity that was dispensable. In contrast, UV light-damaged F plasmid produced an SOS signal independently of the activity of the RecBC enzyme and of the expression of the mini-F, -H, and -G proteins. Our findings are consistent with a picture in which the SOS signal is constituted by stretches of single-stranded DNA on a replicon. Moreover, our present data combined with other data previously published lead to the hypothesis that the SOS signal induced by mini-F plasmid is located in trans on the host chromosome, whereas the one generated by UV light-damaged F plasmid is in cis on the transferred DNA.

Chromosomes, Bacterial↗
PubMed1989

Transformation of Tetrahymena thermophila with a mutated circular ribosomal DNA plasmid vector.

A circular plasmid containing a complete Tetrahymena thermophila rRNA gene (rDNA), with a tandem repeat of a 1.9-kilobase-pair segment encompassing the replication origin and the rRNA promoter, and a polylinker in the 3' nontranscribed spacer, was used to transform T. thermophila by microinjection. Most (20/21) stable transformants contained only recombinant linear palindromic rDNA molecules carrying rDNA sequences from both the donor plasmid and the recipient cell, as shown previously. However, in one transformant, the circular plasmid initially outreplicated the endogenous rDNA and was the major rDNA form for up to 65 generations. Stable circular replicons have not been reported previously in Tetrahymena. A single point mutation (+G) was identified in the repeated promoter of the plasmid maintained in this transformant. After recovery from the Tetrahymena transformant and recloning in Escherichia coli, the mutated circular plasmid again transformed Tetrahymena with stable maintenance of the circular rDNA plasmid. Transformants containing circular replicons were also obtained by using a similar plasmid from which the repeated promoter, but not the repeated replication origin, had been removed by BAL-31 deletion. We therefore propose that repeated rRNA promoters are deleterious in vivo in Tetrahymena, which normally lacks them. Transformants were obtained in 2-5 days compared with the 7-14 days required for transformation with unmutated rDNA plasmids by recombination. Similar results were obtained when a 550-base-pair segment containing the telomerase RNA gene of T. thermophila was inserted in the polylinker of the plasmid. We suggest that this plasmid is a useful vector system for transformation of Tetrahymena.

Animals↗
PubMed1990

Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs.

The development of a bacteriophage P1 cloning system capable of accepting DNA fragments as large as 100 kilobase pairs (kbp) is described. The vectors used in this system contain a P1 packaging site (pac) to package vector and cloned DNA into phage particles, two P1 loxP recombination sites to cyclize the packaged DNA once it has been injected into a strain of Escherichia coli containing the P1 Cre recombinase, a kanr gene to select bacterial clones containing the cyclized DNA, a P1 plasmid replicon to stably maintain that DNA in E. coli at one copy per cell chromosome, and a lac promoter-regulated P1 lytic replicon to amplify the DNA before it is reisolated. An essential feature of the cloning system is a two-stage in vitro packaging reaction that packages vector DNA containing cloned inserts into phage particles that can deliver their DNA to E. coli with near unit efficiency. The packaging reaction can generate 10(5) clones with high molecular weight DNA inserts per microgram of vector DNA. Using NotI fragments from E. coli DNA, it was shown that the system can clone 95- and 100-kbp fragments but not a 106-kbp fragment. Presumably, the combined size of the latter fragment and the vector DNA (13 kbp) exceeds the headful capacity of P1.

Base Composition↗
PubMed1990

Alpha and beta replication origins of plasmid R6K show similar distortions of the DNA helix in vivo.

Plasmid R6K contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. These long inverted repeats (LIRs) occur within the alpha and beta origins of replication and are essential for origin function. In this study, primer-extension analysis of DNA modified in vivo by dimethyl sulfate or KMnO4 revealed that both alpha and beta LIRs acquire similar structural distortions of the DNA helix in a functional R6K replicon. These distortions were not seen in plasmids containing isolated LIR sequences. In the functional replicon, the dimethyl sulfate and KMnO4 hyperreactive sites appear on complementary strands and are located to one side of an internal palindromic sequence within the LIRs. This asymmetry coincides with the primary direction of DNA replication from alpha and beta origins in vivo. We also observed two intermediate structures when certain R6K cis- or trans-acting elements are missing. Sequences near the alpha origin are required for generation of the dimethyl sulfate hyperreactive sites, whereas sequences near the beta origin are responsible for the appearance of KMnO4 hyperreactive sites. We suggest that these structures represent a hierarchy that leads to a "locked" preinitiation complex, which functions to synchronize and determine the direction of replication from the alpha and beta replication origins in vivo.

Base Sequence↗
PubMed1992

A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy.

The bacteriophage P1 cloning system can package and propagate DNA inserts that are up to 95 kilobases. Clones are maintained in Escherichia coli by a low-copy replicon in the P1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-D-thiogalactopyranoside. To overcome the necessity of screening clones for DNA inserts, we have developed a P1 vector with a positive selection system that is based on the properties of the sacB gene from Bacillus amyloliquefaciens. Expression of that gene kills E. coli cells that are grown in the presence of sucrose. In the new P1 vector (pAd10sacBII) sacB expression is regulated by a synthetic E. coli promoter that also contains a P1 C1 repressor binding site. A unique BamHI cloning site is located between the promoter and the sacB structural gene. Cloning DNA fragments into the BamHI site interrupts sacB expression and permits growth of plasmid-containing cells in the presence of sucrose. We have also bordered the BamHI site with unique rare-cutting restriction sites Not I, Sal I, and Sfi I and with T7 and Sp6 promoter sequences to facilitate characterization and analysis of P1 clones. We describe here the use of Not I digestion to size the cloned DNA fragments and RNA probes to identify the ends of those fragments. The positive selection P1 vector provides a 65- to 75-fold discrimination of P1 clones that contain inserts from those that do not. It therefore permits generation of genomic libraries that are much easier to use for gene isolation and genome mapping than are our previous libraries. Also, the new vector makes it feasible to generate P1 libraries from small amounts of genomic insert DNA, such as from sorted chromosomes.

Animals↗
PubMed1997

A variable domain of delayed replication in FRAXA fragile X chromosomes: X inactivation-like spread of late replication.

The timing of DNA replication in the Xq27 portion of the human X chromosome was studied in cells derived from normal and fragile X males to further characterize the replication delay on fragile X chromosomes. By examining a number of sequence-tagged sites (STSs) that span several megabases of Xq27, we found this portion of the normal active X chromosome to be composed of two large zones with different replication times in fibroblasts, lymphocytes, and lymphoblastoid cells. The centromere-proximal zone replicates very late in S, whereas the distal zone normally replicates somewhat earlier and contains FMR1, the gene responsible for fragile X syndrome when mutated. Our analysis of the region of delayed replication in fragile X cells indicates that it extends at least 400 kb 5' of FMR1 and appears to merge with the normal zone of very late replication in proximal Xq27. The distal border of delayed replication varies among different fragile X males, thereby defining three replicon-sized domains that can be affected in fragile X syndrome. The distal boundary of the largest region of delayed replication is located between 350 and 600 kb 3' of FMR1. This example of variable spreading of late replication into multiple replicons in fragile X provides a model for the spread of inactivation associated with position-effect variegation or X chromosome inactivation.

Cell Cycle↗
PubMed2002

Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.

Cell Line↗
PubMed2002

Interfacial domains in Sindbis virus 6K protein. Detection and functional characterization.

Alphavirus 6K is a short, constitutive membrane protein involved in virus glycoprotein processing, membrane permeabilization, and the budding of virus particles. The amino-terminal region that immediately precedes the transmembrane anchor contains a conserved sequence motif consisting of two interfacial domains separated by Asn and Gln residues. The presence of this motif confers on the 6K pretransmembrane region the tendency to partition into the membrane interface. To study the functional importance of the interfacial sequences, three different Sindbis virus 6K variants were obtained with the following modifications: 9YLW11xAAA, 18FWV20xAAA, and 9YLW11xAAA/18FWV20xAAA. Reconstituted mutant viruses were infectious and showed no defects in glycoprotein processing, although virus budding was hampered. Single 6K expression in Escherichia coli cells showed interfacial mutants to have a diminished capacity to modify membrane permeability and to have lower toxicity. In particular, the 9YLW11xAAA/18FWV20xAAA variant was expressed at high levels and did not enhance membrane permeability significantly, although it retained its integral membrane protein condition. Parallel analyses of membrane permeabilization in baby hamster kidney cells were carried out using a Sindbis virus replicon that synthesized both capsid protein and 6K. Transfection of the construct with wild-type 6K strongly increased permeability to the antibiotic hygromycin B. Replicons encoding 6K interfacial mutants induced lower membrane permeabilization. Again, the greatest impairment was observed for the 9YLW11xAAA/18FWV20xAAA variant, permeabilization activity of which was approximately 10% that of wild-type 6K. These findings show the importance of the interfacial 6K sequence for virus budding and modification of membrane permeability.

Amino Acid Sequence↗
PubMed2003

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

Adenosine↗
PubMed2003

Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro.

The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2'-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-alpha results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2'-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2'-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections.

Animals↗
PubMed2004

Hepatitis C virus suppresses the IRE1-XBP1 pathway of the unfolded protein response.

Hepatitis C virus (HCV) gene expression disrupts normal endoplasmic reticulum (ER) functions and induces ER stress. ER stress results from the accumulation of unfolded or misfolded proteins in the ER; cells can alleviate this stress by degrading or refolding these proteins. The IRE1-XBP1 pathway directs both protein refolding and degradation in response to ER stress. Like IRE1-XBP1, other branches of the ER stress response mediate protein refolding. However, IRE1-XBP1 can also specifically activate protein degradation. We show here that XBP1 expression is elevated in cells carrying HCV subgenomic replicons, but XBP1 trans-activating activity is repressed. This prevents the IRE1-XBP1 transcriptional induction of EDEM (ER degradation-enhancing alpha-mannosidase-like protein). The mRNA expression of EDEM is required for the degradation of misfolded proteins. Consequently, misfolded proteins are stable in cells expressing HCV replicons. HCV may suppress the IRE1-XBP1 pathway to stimulate the synthesis of its viral proteins. IRE1alpha-null MEFs, a cell line with a defective IRE1-XBP1 pathway, show elevated levels of HCV IRES-mediated translation. Therefore, HCV may suppress the IRE1-XBP1 pathway to not only promote HCV expression but also to contribute to the persistence of the virus in infected hepatocytes.

Animals↗