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At least 469 records · Page 26Linked to original sources
PubMed1993

[Parallel import of drug preparations. A study of price development of parallel import preparations versus original preparations].

Price developments for the first parallel imported preparations and the corresponding original preparations have been followed over the period 1st January 1992 to 1st June 1992 with the aim of registering any possible effects of the parallel import on price levels. Altogether 174 parallel imported preparations--including different strengths and administration forms--received marketing permission in the period, and by 1st June 1992 74 parallel imported preparations had come on the market. The main part of the parallel imported preparations are marketed at a lower price than the original preparations. At the end of the investigation period, 56% of the marketed parallel imported preparations were being sold at a price that was on average 5-10% below that of the corresponding original preparation; 24% were being sold at a price 0-4% below the original and 20% were being sold at more than 10% below the original preparations. The effect on the prices of the original preparations has been variable. In the beginning of the study period we saw four examples of large reductions in the price of original preparations (up to 45%) after the parallel imports began to be marketed, but at the end of the study period the marketing of the cheaper preparations seemed to have no effect on the prices of the original preparations, which remained unchanged.

Denmark↗
PubMed1993

DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication.

The ATP-dependent binding of simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication is an essential step in the initiation of SV40 DNA synthesis. Previous studies indicated that the ATP-dependent complex consists of a double hexamer of T antigen at the origin. The binding reaction and the subsequent unwinding of the duplex DNA from the origin were examined using a gel mobility shift assay. T antigen bound to the core origin cooperatively in the presence of ATP. In the presence of human single-stranded DNA-binding protein (HSSB), T antigen, complexed to the core origin, started the unwinding of duplex DNA. At low concentrations of T antigen and in the presence of ATP, DNA polymerase alpha (pol alpha) stimulated the binding of T antigen to the core origin, while HSSB did not. This stimulation resulted in an increase in the subsequent unwinding reaction in the presence of HSSB. Primase alone did not affect the binding reaction and was not required for the stimulation by pol alpha. The stimulation required the hydrolysis of ATP and the AT tract domain of the core origin. Kinetic studies showed that while pol alpha stimulated the binding of T antigen to the core origin, it did not stabilize the complex. Pol alpha also stimulated the formation of the ATP-dependent T antigen-site I complex, a region that also contains an AT-rich sequence. These observations imply a regulatory role for pol alpha in the initiation of SV40 DNA replication.

Adenosine Triphosphate↗
PubMed1996

The origin of IgG production and homogeneous IgG components after allogeneic bone marrow transplantation.

Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G1m and G2m allotype levels were quantified in sera obtained at regular intervals between 3 months and 5 years after BM transplantation (BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, IgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the origin of total IgG production, the origin of homogeneous IgG components (H-IgG) appearing after BMT was investigated. H-IgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin were present in 8 of 13 informative recipients for a period of up to 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number of graft recipients. It is our hypothesis that lack of graft-versus-host disease (GVHD) in the majority of these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients who received allogeneic BMT for the treatment of multiple myeloma.

Anemia, Aplastic↗
PubMed1995

Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC.

Characterization of the proteins that interact with replication origins, as well as characterization of the mechanisms by which the levels and activities of these proteins are regulated during the cell cycle, is required to understand the initiation of chromosomal DNA replication in eukaryotic cells. We have previously shown that the first detectable step in the assembly of initiation complexes in vivo involves the binding of the multisubunit origin recognition complex (ORC) and the general transcription/replication factor ABF1 protein to origins. In this paper we show that ORC is present in cells at low levels, corresponding to little more than one complete complex per replication origin, indicating that in vivo origin recognition by ORC is extremely efficient. We show that this efficient recognition requires two sequence elements, the essential A element containing the ARS consensus sequence and the functionally important B1 element, both in vitro and in vivo. Moreover, we show that origin binding by ORC in vivo does not require any other functional sequence element, indicating that it occurs independently of the binding of other factors, such as ABF1. Our results suggest a model for the roles of the individual elements of yeast replication origins.

Base Sequence↗
PubMed2004

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus.

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.

Archaeal Proteins↗
PubMed2004

Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus.

Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix-turn-helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.

Binding Sites↗
PubMed2001

DNA replication forks pause at silent origins near the HML locus in budding yeast.

Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival at HML led to the elimination of the pause sites at the HML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed.

DNA Replication↗
PubMed2000

Circular YAC vectors containing short mammalian origin sequences are maintained under selection as HeLa episomes.

pYACneo, a 15.8-kb plasmid, contains a bacterial origin, G418-resistance gene, and yeast ARS, CEN, and TEL elements. Three mammalian origins have been cloned into this circular vector: 343, a 448-bp chromosomal origin from a transcribed region of human chromosome 6q; X24, a 4.3-kb element containing the hamster DHFR origin of bidirectional replication (oribeta), and S3, a 1.1-kb human anti-cruciform purified autonomously replicating sequence. The resulting constructs have been transfected into HeLa cells, and G418-resistant subcultures were isolated. The frequency of G418-resistant transformation was 1.7-8.7 times higher with origin-containing YACneo than with vector alone. After >45 generations under G418 selection, the presence of episomal versus integrated constructs was assessed by fluctuation assay and by PCR of supercoiled, circular, and linear genomic cellular DNAs separated on ethidium bromide-cesium chloride gradients. In stable G418-resistant subcultures transfected with vector alone or with linearized constructs, as well as in some subcultures transfected with circular origin-containing constructs, resistance was conferred by integration into the host genome. However, several examples were found of G418-resistant transfectants maintaining the Y.343 and the YAC.S3 circular constructs in a strictly episomal state after long-term culture in selective medium, with 80-90% stability per cell division. The episomes were found to replicate semiconservatively in a bromodeoxyuridine pulse-labeling assay for </=130 cell generations after transfection. Furthermore, after </=172 cell generations rescued episomal DNA could be isolated intact and unrearranged, and could be used to retransform bacteria. These versatile constructs, containing mammalian origins, have the capacity for further modification with human telomere or large putative centromere elements, in an effort to move towards construction of a human artificial chromosome.

Bromodeoxyuridine↗
PubMed1999

Replication origins in metazoan chromosomes: fact or fiction?

The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development.

Animals↗
PubMed2005

Transcription factors and DNA replication origin selection.

The chromosomes of eukaryotic cells possess many potential DNA replication origins, of which a subset is selected in response to the cellular environment, such as the developmental stage, to act as active replication start sites. The mechanism of origin selection is not yet fully understood. In this review, we summarize recent observations regarding replication origins and initiator proteins in various organisms. These studies suggest that the DNA-binding specificities of the initiator proteins that bind to the replication origins and promote DNA replication are primarily responsible for origin selection. We particularly focus on the importance of transcription factors in the origin selection process. We propose that transcription factors are general regulators of the formation of functional complexes on the chromosome, including the replication initiation complex. We discuss the possible mechanisms by which transcription factors influence the selection of particular origins.

Animals↗
PubMed2003

Same origins of DNA replication function on the active and inactive human X chromosomes.

We previously characterized a functional origin of DNA replication at the transcriptional promoter of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Cohen et al. [2002] J. Cell. Biochem. 85:346-356). This origin was mapped using a quantitative PCR assay to evaluate the relative abundance of HPRT markers in short nascent DNA strands isolated from asynchronous cultures of male fibroblasts. The HPRT gene on the X chromosome is transcriptionally active in male human fibroblasts. It is known that on the heterochromatic X chromosome in female cells the HPRT gene is transcriptionally silenced and its replication timing changes from early to late in S phase. This change in replication timing could indicate that replication of the HPRT gene is under the control of different origins of DNA replication in the active (euchromatic, early replicating) and the inactive (heterochromatic, late replicating) X chromosomes. In the present study, we identified the location of the origin of replication of a second X chromosome gene, glucose-6-phosphate dehydrogenase (G6PD), which we mapped to its transcriptional promoter, in normal male human fibroblasts. Then, we determined the activity of the previously identified HPRT and the G6PD human origins in hybrid hamster cells carrying either the active or the inactive human X chromosome. The results of these studies clearly demonstrated that the human HPRT and G6PD origins of replication were utilized to the same extent in the active and the inactive X chromosomes. Therefore, transcription activity at the HPRT and G6PD genes is not necessary for initiation of DNA replication at the origins mapped to these chromosomal loci.

Animals↗
PubMed1997

RNA-DNA hybrid formation at a bacteriophage T4 replication origin.

The bacteriophage T4 replication origins ori(uvsY) and ori(34) each contain two distinct components: a T4 middle-mode promoter that is strictly required for replication and a downstream region of about 50 bp that is required for maximal levels of replication. Here, we present evidence that structure of the downstream region is important for replication initiation. Based on sensitivity to a single-stranded DNA-specific nuclease in vitro the downstream region behaves as a DNA unwinding element. The propensity to unwind is probably important for origin activity in vivo, because replication activity is maintained when the native downstream region is replaced with a heterologous DNA unwinding element from pBR322 in either orientation. We analyzed the origin DNA for possible unwinding in vivo by using potassium permanganate, a chemical that reacts with unpaired pyrimidine bases. The non-template strand, but not the template strand, became hypersensitive to permanganate after T4 infection regardless of whether replication could occur. Strand-specific permanganate hypersensitivity was also observed in artificial origins containing the pBR322 DNA unwinding element in either orientation. Hypersensitivity was only detected when the origin contained a promoter that would be active during T4 infection. Furthermore, the origin transcript itself appears to be necessary for hypersensitivity since insertion of a transcriptional terminator abolishes hypersensitivity downstream of the termination site. Our results strongly suggest that the downstream region functions as a DNA unwinding element during replication initiation, leading to the formation of a persistent RNA-DNA hybrid at the origin.

Bacteriophage T4↗
PubMed1997

Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction.

A procedure was developed for mapping origins of DNA replication in mammalian cell chromosomes based on determining the relative abundance of nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from recently activated origins and the quantification, within this sample, of the relative abundances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin mapping procedure (i) allows analysis of single-copy genomic regions, (ii) can be performed on cultured and primary cells in the absence of any chemical treatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of the cis- and trans-acting elements required for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an approximately 500-bp segment in the human lamin B2 gene domain and within an approximately 800-bp segment in the hamster dihydrofolate reductase gene locus.

Animals↗
PubMed1997

Origin-specific initiation of mammalian nuclear DNA replication in a Xenopus cell-free system.

The introduction of Chinese hamster ovary (CHO) cell nuclei into Xenopus egg extracts provides the only cell-free system that can efficiently initiate replication at a specific metazoan replication origin. With intact late-G1-phase nuclei as a substrate, the pattern of initiation sites for replication at the CHO dihydrofolate reductase (DHFR) locus is indistinguishable from that observed in cultured cells. By contrast, with early-G1-phase nuclei or with late-G1-phase nuclei that have damaged nuclear envelopes, these same extracts efficiently initiate replication at apparently random sites. Thus, at a distinct point during G1 phase [origin decision point (ODP)], nuclei experience a transition that is required for specific recognition of the DHFR origin by Xenopus egg cytosol. Described here are the basic requirements to achieve origin-specific initiation, which include: 1) a cell line that can be synchronized in G1 phase, 2) a method to prepare intact nuclei, 3) a technique to map origins with a few million cells, and 4) a small colony of Xenopus laevis. Immunodepletion of specific gene products allows one to test hypotheses about the requirements for origin recognition. Here we show that depletion of the Xenopus origin recognition complex subunit XORC2 from Xenopus egg extracts has no influence on the efficiency of replication or the pattern of initiation sites with either pre-ODP or post-ODP nuclei.

Animals↗
PubMed1999

Chorion gene amplification in Drosophila: A model for metazoan origins of DNA replication and S-phase control.

The mechanisms controlling duplication of the metazoan genome are only beginning to be understood. It is still unclear what organization of DNA sequences constitutes a chromosomal origin of DNA replication, and the regulation of origin activity during the cell cycle has not been fully revealed. We review recent results that indicate that chorion gene amplification in follicle cells of the Drosophila ovary is a model for investigating metazoan replication. Evaluation of cis sequence organization and function suggests that chorion loci share attributes with other replicons and provides insights into metazoan origin structure. Moreover, recent results indicate that chorion origins respond to S-phase control, but escape mechanisms that inhibit other origins from firing more than once in a cell cycle. Several identified genes that mediate amplification are critical for the cell cycle control of replication initiation. It is likely that further genetic screens for mutations that disrupt amplification will identify the cadre of proteins associated with origins and the regulatory pathways that control their activity. Furthermore, the recent development of methods to detect amplification in situ has uncovered new aspects of its developmental control. Examining this control will reveal links between developmental pathways and the cell cycle machinery. Visualization of amplifying chorion genes with high resolution also represents an opportunity to evaluate the influence of nuclear and chromosome structure on origin activity. The study of chorion amplification in Drosophila, therefore, provides great potential for the genetic and molecular dissection of metazoan replication.

Animals↗
PubMed2004

Identification of replication origins in the genome of the methanogenic archaeon, Methanocaldococcus jannaschii.

Methanocaldococcus jannaschii has been notorious as an archaeon in which the replication origins are difficult to identify. Although extensive efforts have been exerted on this issue, the locations of replication origins still remain elusive 7 years after the publication of its complete genome sequence in 1996. Ambiguous results were obtained in identifying the replication origins of M. jannaschii based on all theoretical and experimental approaches. In the genome of M. jannaschii, we found that an ORF (MJ0774), annotated as a hypothetical protein, is a homologue of the Cdc6 protein. The position of the gene is at a global minimum of the x component of the Z curve, i.e., RY disparity curve, which has been used to identify replication origins in other Archaea. In addition, an intergenic region (694,540-695,226 bp) that is between the cdc6 gene and an adjacent ORF shows almost all the characteristics of known replication origins, i.e., it is highly rich in AT composition (80%) and contains multiple copies of repeat elements and AT stretches. Therefore, these lines of evidence strongly suggest that the identified region is a replication origin, which is designated as oriC1. The analysis of the y component of the Z curve, i.e., MK disparity curve, suggests the presence of another replication origin corresponding to one of the peaks in the MK disparity curve at around 1,388 kb of the genome.

Amino Acid Sequence↗
PubMed2004

Multiple origins of replication in archaea.

Until recently, the only archaeon for which a bona fide origin of replication was reported was Pyrococcus abyssi, where a single origin was identified. Although several in silico analyses have suggested that some archaeal species might contain more than one origin, this has only been demonstrated recently. Two studies have shown that multiple origins of replication function in two archaeal species. One study identified two origins of replication in the archaeon Sulfolobus solfataricus, whereas a second study used a different technique to show that both S. solfataricus and Sulfolobus acidocaldarius have three functional origins. These are the first reports of archaea having multiple origins. This finding has implications for research on the mechanisms of DNA replication and evolution.

Archaea↗
PubMed1996

Large, complex modular structure of a fission yeast DNA replication origin.

BACKGROUND: In the budding yeast, Saccharomyces cerevisiae, each DNA replication origin is associated with an autonomously replicating sequence (ARS) element. Each element contains several modules, including an essential close match to the 11 base-pair (bp) ARS consensus sequence (ACS) and two or three short (< 20 bp) stimulatory motifs, within a stretch of approximately 150 bp or less. To determine whether a similar origin structure exists in the evolutionarily distant fission yeast, Schizosaccharomyces pombe, we used deletion and linker substitution scanning to identify the sequences important for the function of ars3002, a chromosomal replication origin. RESULTS: We detected two large (30-55 bp) essential regions and several additional stimulatory sequences within a 600 bp stretch of a restriction fragment containing ars3002. The two essential regions are similar to each other, and sequences similar to them are found in all known S. pombe ARS elements, suggesting that one or both of them may represent the S. pombe equivalent of the S. cerevisiae ACS. CONCLUSIONS: Like S. cerevisiae origins, the S. pombe origin, ars3002, possesses a modular structure, but the number and size of modules is greater for ars3002, and ars3002 is larger than S. cerevisiae origins. These observations suggest that origin function in S. pombe requires more protein-DNA interactions than in S. cerevisiae.

Base Sequence↗