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At least 1,405 records · Page 78Linked to original sources
PubMed1990

Novel cloning vectors for Bacillus thuringiensis.

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.

Bacillus thuringiensis↗
PubMed1985

The herpes simplex virus amplicon: analyses of cis-acting replication functions.

Previous studies have shown that defective virus vectors (amplicons) derived from herpes simplex viruses could be efficiently propagated in virus stocks in the presence of trans-acting helper virus functions. The present study established that two separate cis-acting functions--a DNA replication origin and a cleavage/packaging signal--are required for amplicon propagation. Using deleted derivatives of cloned amplicons, we mapped one of the viral DNA replication origins (ori-2 or oriL) at coordinate 0.422 of the standard HSV-1 genome and at an equivalent position within the HSV-2 genome.

Base Sequence↗
PubMed1992

Activation of a mammalian origin of replication by chromosomal rearrangement.

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

Animals↗
PubMed2001

Initiating DNA synthesis: from recruiting to activating the MCM complex.

The exact duplication of a genome once per cell division is required of every proliferating cell. To achieve this goal, eukaryotes adopt a strategy that limits every replication origin to a single initiation event within a narrow window of the cell cycle by temporally separating the assembly of the pre-replication complex (pre-RC) from the initiation of DNA synthesis. A key component of the pre-RC is the hexameric MCM complex, which is also the presumed helicase of the growing forks. An elaborate mechanism recruits the MCM complex to replication origins, and a regulatory chain reaction converts the poised, but inactive, MCM complex into an enzymatically active helicase. A growing list of proteins, including Mcm10 and Cdt1, are involved in the recruitment process. Two protein kinases, the Cdc7-Dbf4 kinase (DDK) and the cyclin-dependent kinase (CDK), trigger a chain reaction that results in the phosphorylation of the MCM complex and finally in the initiation of DNA synthesis. A composite picture from recent studies suggests that DDK is recruited to the pre-RC during G1 phase but must wait until S phase to phosphorylate the MCM complex. CDK is required for the recruitment of Cdc45 and other downstream components of the elongation machinery.

Animals↗
PubMed1990

Mutational and physiological analyses of plasmid pT181 functions expressing incompatibility.

Plasmid pT181 is a small multicopy plasmid from Staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, Inc3A and Inc3B. Inc3A incompatibility is expressed by the primary replication control determinant, copA, which specifies two small transcripts, RNA I and RNA II, that jointly inhibit the synthesis of the rate-limiting initiator protein, RepC. Inc3B incompatibility is expressed by the leading strand replication origin and is due to competition for RepC. The copA region from each of 11 different pT181 copy number mutants was cloned onto the pT181-compatible vector, pE194, and tested for its ability to inhibit the replication of pT181 and its copy number mutants. The pT181 replication origin was also cloned and tested for its ability to inhibit the replication of the same plasmids. In general copA mutations that alter the production or sequence of RNA I and RNA II greatly reduced or completely eliminated Inc3A activity. Unlike the wild-type, all of the copy mutants were resistant to Inc3B inhibition. The separately cloned wild-type copA and ori regions each reduced the copy number of pT181 in proportion to their gene dosage, but neither blocked replication completely. It is proposed that the cloned Inc determinants cause incompatibility by interfering with the plasmid's copy correction mechanism; this interference destabilizes the plasmid even under conditions where its average copy number is not greatly reduced.

Bacterial Proteins↗
PubMed1994

A versatile plasmid vector system for the regulated expression of genes in Escherichia coli.

A series of plasmid expression vectors, which support the regulated and efficient expression of genes in Escherichia coli, have been constructed. The vectors consist of a DNA replication origin cassette, a promoter cassette, an efficient ribosome binding site together with a polylinker region and a lacZ gene. Several types of replication origins and promoter sequences are each available on cassettes. Fusion of the 5' TG dinucleotide of the gene under consideration to the A nucleotide, present on the vector, results in an ATG start codon and allows, in combination with the plasmid-borne ribosome binding site, the efficient expression of the cloned gene. Additionally, a second fusion of the gene at its 3' end with the lacZ gene, which is available in all three reading frames relative to the polylinker region, allows rapid selection of the correctly fused genes. As an example of the cloning of a regulatory gene, this vector system was used for the expression of the dnaA gene, of Escherichia coli, the initiator protein for DNA replication.

Bacterial Proteins↗
PubMed1999

Activation of DNA replication in yeast by recruitment of the RNA polymerase II transcription complex.

Activators of transcription are known to also play an important and direct role in activating DNA replication. However, the mechanism whereby they stimulate replication has remained elusive. One model suggests that, in the context of replication origins, transcriptional activators work by interacting with replication factors. We show that a defined, single interaction between a DNA-bound derivative of the activator Gal4 and Gal11P, a mutant form of the RNA polymerase II holoenzyme component Gal11, suffices for stimulating DNA replication as it does for transcription. Moreover, recruitment of TBP, which can activate transcription from a gene promoter, also stimulates DNA replication from an origin site. These results strongly argue that transcriptional activators may not necessarily need to contact DNA replication factors directly, but can stimulate replication by recruiting the RNA polymerase II transcription complex to DNA.

DNA Replication↗
PubMed1984

Dual-origin plasmid vectors whose origin of replication is controlled by the coliphage lambda promoter pL.

The insertion of an XhoI linker 30 bp from the 5' end of the RNA II primer for ColE1 plasmid replication has allowed us to replace the natural RNA II promoter by other controllable promoters, in particular lambda pL. Manipulation of this modified origin was facilitated by constructing dual-origin plasmids, stable maintenance being directed by the pSC101 origin. Experiments showed that such dual-origin plasmids could be stably maintained at approximately four copies per chromosome at 30 degrees C and readily amplified by thermal induction of strains carrying a thermolabile lambda repressor. The use of such plasmids for the construction of stable, amplifiable expression vectors is discussed.

Bacteriocin Plasmids↗
PubMed2002

Mapping of an origin of DNA replication near the transcriptional promoter of the human HPRT gene.

A quantitative PCR method was used to map a functional origin of DNA replication in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. This PCR method measures the abundance of specific sequences in short fragments of newly replicated DNA from logarithmically growing cells. Quantitative measurements rely on synthetic molecules (competitors) that amplify with the same primer sets as the target molecules, but generate products of different sizes. This method was first utilized to determine the position of the replication origin near the lamin B2 gene (Giacca et al. [1994] Proc. Natl. Acad. Sci. U S A. 91:7119-7123). In the present study, primer sets were tested along a 16-kb region near exon 1 of the HPRT gene. The most abundant fragment was found to be located in the first intron of HPRT, just downstream of the promoter and exon 1 of the gene, and approximately 3.5 kb upstream of a previously reported autonomously replicating sequence (Sykes et al. [1988] Mol. Gen. Genet. 212:301-309).

Chromosome Mapping↗
PubMed1990

Effect of human cytomegalovirus on replication of SV40 origin and the expression of T antigen.

Human cytomegalovirus (HCMV) induces replication of the cloned SV40 origin of DNA replication in both productive and nonproductive infections. HCMV-induced replication of the SV40 DNA origin required the presence of T antigen. Human embryonic lung (HEL) cells were found to be fully permissive for SV40 origin replication only in the presence of HCMV gene expression. In addition, expression of plasmid encoded SV40 T antigen in HEL cells was only induced in the presence of HCMV. Cotransfection of the SV40-cloned origin of replication and seven overlapping cosmids containing the entire HCMV genome is capable of stimulating replication of the SV40 origin. We conclude that induction of the SV40 origin of replication by HCMV is (i) not due to any component of the HCMV virion, (ii) has a requirement for T antigen expression, (iii) occurs in an SV40 nonproductive cell, when T antigen expression is induced by HCMV gene expression.

Animals↗
PubMed1991

Origins of replication: timing and chromosomal position.

Several new methods have been used to localize replication initiation sites in mammalian chromosomes. The results of these studies argue strongly for the presence of defined sequence elements that function much like the origins in the genomes of simple microorganisms. However, relatively disparate results from in vivo and in vitro studies suggest that initiation reactions in mammalian chromosomes may have unique features, possibly related to a more complicated chromosomal architecture.

Animals↗
PubMed1994

Analysis of cis and trans acting elements required for the initiation of DNA replication in the Bacillus subtilis bacteriophage SPP1.

The development of SPP1 has been studied in several B. subtilis mutants conditionally defective in initiation of DNA replication. Initiation of SPP1 replication is independent of the host DnaA (replisome organizer), DnaB, DnaC and DnaI products, but requires the DnaG (DNA primase) and the DNA gyrase. Furthermore, SPP1 replication is independent of the DnaK (heat shock) protein. The phage-encoded products required for initiation of SPP1 replication have been genetically characterized. Analysis of the nucleotide sequence (3.292 kilobases) of the region where SPP1 initiation replication mutants map, revealed five open reading frames (orf). We have assigned genes 38, 39 and 40 to three of these orfs, which have the successive order gene 38-gene 39-orf39,1-gene 40-orf41. The direction of transcription of the reading frames, the lengths of the mRNAs as well as the transcription start point, upstream of gene 38 (PE2), were identified. Proteins of 29.9, 14.6 and 46.6 kDa were anticipated from translation of gene 38, gene 39 and gene 40, respectively. The purified G38P and G39P have estimated molecular masses of 31 and 15 kDa. G38P and G39P do not share significant identity with primary protein sequences currently available in protein databases, whereas G40P shares substantial homology with a family of DNA primase-associated DNA helicases. G38P binds specifically to two discrete SPP1 DNA restriction fragments (EcoRI-4 and EcoRI-3). The G38P binding site on EcoRI-4 was localized on a 393 bp DNA segment, which lies within the coding sequence of gene 38. The putative binding site on EcoRI-3 was inferred by DNA sequence homology, it maps in a non-coding segment. G39P, which does not bind to DNA, is able to form a complex with G38P. The organization of the SPP1 genes in the gene 38 to gene 40 interval resembles that one found in the replication origin regions of different Escherichia coli double-stranded DNA phages (lambda, phi 80 and P22). We propose that the conserved gene organization is representative of the replication origin region of a primordial phage.

Amino Acid Sequence↗
PubMed2000

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication.

During late mitosis and early G(1), a series of proteins are assembled onto replication origins, resulting in them becoming 'licensed' for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.

Animals↗
PubMed2001

Analysis of pFQ31, a 8551-bp cryptic plasmid from the symbiotic nitrogen-fixing actinomycete Frankia.

The actinomycete Frankia has never been transformed genetically. To favour the development of Frankia cloning vectors, we have fully sequenced the Frankia alni pFQ31 cryptic plasmid and performed analyses to characterise its coding and non-coding regions. This plasmid is 8551 bp-long and contains 72% G+C. Computer-assisted analyses identified 18 open reading frames (ORFs). These ORFs show a synonymous codon usage different from the one of Frankia chromosomal genes, suggesting an evolutionary bias linked to the nature of the replicon or a horizontal transfer. Three ORFs were found to encode genes likely to be involved in plasmid replication and stability: parFA (partition protein), ptrFA (transcriptional repressor of the GntR family) and repFA (initiation of replication). DNA signatures of a replication origin were identified in the ptrFA-repFA intergenic region. These structural motifs are similar to those observed among origins of iteron-containing plasmids replicating via a θ mode.

Actinomycetales↗
PubMed2001

Epstein-Barr virus nuclear antigen-1-dependent and -independent oriP-binding cellular proteins.

OBJECTIVE: Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) and the replication origin, oriP, are essential for the replication and maintenance of latent EBV DNA in cells, but no enzymatic activity has been associated with EBNA-1 protein alone. In this study, we have searched for host cellular proteins that interact with EBNA-1 protein in various B cell lines latently infected with EBV, including a recently EBV growth-transformed cell line. METHODS: By using gel shift analysis, we investigated the interactions of an oligonucleotide containing a single EBNA-1 recognition site, derived from the family of repeats (FR) element of oriP, with protein from cell extracts. RESULTS: The FR oligonucleotide bound a (72-kD) cellular protein in the absence of EBNA-1 and without induction of the previously reported 'anti-EBNA-1 proteins'. The FR oligonucleotide formed complexes with additional proteins from EBNA-1-synthesizing cell lines; these complexes were abolished or supershifted by anti-EBNA-1 monoclonal antibodies. SDS-PAGE analyses of 35S-Met-labeled proteins that bound to a biotin- conjugated FR oligonucleotide, fractionated by a glycerol gradient centrifugation and affinity-purified with streptavidin, showed three major bands, a 72-kD protein, the FR binding of which seemed to be independent of EBNA-1, a 64-kD protein in both EBNA-1-transfected and latently EBV-infected cell lines, and a 45-kD protein in EBV-infected cell lines, which was most prominent in a recently EBV growth-transformed cell line. CONCLUSIONS: The FR element forms complexes with cellular proteins in the absence and presence of EBNA-1. These 72-, 64- and 45-kD cellular proteins might be involved in the function of the oriP and EBNA-1 system.

Binding Sites↗
PubMed2025

Nanopore-based sequencing of active DNA replication reveals key principles of metazoan replication fork progression, origin and termination sites.

Balancing replication fork progression and origin usage is essential to maintain genome stability, but measuring replication fork progression rates and origin usage throughout the genome has been challenging. Here, we use nanopore sequencing combined with DNAscent to measure replication fork progression together with origin and termination site usage with single-molecule precision throughout the Drosophila genome with nearly full genome coverage. We find that replication fork progression rates are not uniform throughout the genome. Rather, fork progression is slowest in euchromatin, and this is not correlated with active transcription. Replication origins are also influenced by chromatin, but the exact position of initiation is highly variable and are often several kilobases away from ORC binding sites. Termination sites lack any chromatin or sequence motifs and appear nearly random throughout the genome. By measuring DNA replication dynamics at near full genome coverage, our work reveals key principles of metazoan replication dynamics.

Journal Article↗
PubMed1996

Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria.

We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6K gamma DNA replication origin (oriR(R6K gamma) so they replicate only in bacteria supplying the pi replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of pi. They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria. Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA. Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker. One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants. Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones. Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker. Plasmid-free segregants with an allele replacement can be subsequently selected as TetS or SucR recombinants. A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.

Alleles↗
PubMed2005

An oriC-like distribution of GATC sites mediates full sequestration of non-origin sequences in Escherichia coli.

Sequestration of newly replicated origins is one of the mechanisms required to limit initiation of Escherichia coli chromosome replication to once per generation. Origin sequestration lasts for a considerably longer period of time than the sequestration of other newly replicated regions of the chromosome. The reason for this may be the high number of GATC sites present in the origin. Alternatively, other sequence elements in the origin region may be important for its prolonged sequestration. To distinguish between these possibilities we constructed a DNA fragment containing ten GATC sites distributed with the same spacing as the ten GATC sites in the left half of oriC, but with random sequence between the GATC sites, and inserted it at a non-sequestered chromosome location. Sequestration of this GATC-cluster lasted as long as that of oriC, or even longer. The result shows that the presence of ten GATC sites, distributed as in oriC, is sufficient to cause full sequestration, and that other sequence elements most likely do not contribute to sequestration.

Base Sequence↗