SEARCH · PubMed Health

Results for “DNA replication origin”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,423 records · Page 79Linked to original sources

PubMed1999

[Coupling of immunomagnetic separation and polymerase chain reaction for the rapid detection of salmonellae in minced meat and chicken giblets].

Coupling of immunomagnetic separation and polymerase chain reaction was used after preenrichment for the rapid detection of salmonellae in minced meat and chicken giblets. The primers used are specific to the origin of DNA replication (oriC) on the Salmonella chromosome and produce a 163 bp DNA fragment. The method allowed a screening of Salmonella free samples within 24 hours. It detected 5 serovars when inoculated in minced meat at a contamination level of 1 to 4 cfu/g. Compared with the method L 00.00-20 of the Official Collection of Methods of Analysis according to section 35 of the German Law on Food and Commodities it showed a sensitivity of 94% and a specificity of 87.5% for the examination of chicken giblets (n = 45) and a specificity of 100% for the examination of minced meat (n = 63).

Animals↗

PubMed2003

Cellular mobile genetic elements in the regulatory region of the pneumotropic mouse polyomavirus genome: structure and function in viral gene expression and DNA replication.

DNA from the murine pneumotropic virus was extracted from virus in lung tissue of infected mice, and the regulatory region of the genome was amplified by PCR. The regulatory region of individual plasmid cloned DNA molecules appeared to have heterogeneous enhancer segments, whereas the protein-coding part of the genome had a uniform length. Nucleotide sequence analysis revealed that the majority of the DNA molecules had a structure differing from the standard type. A 220-bp insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was prominent. There were two variants of the 220-bp insertion, differing at two nucleotide positions at one of the termini. Other DNA molecules had complete or partial deletions of these structures and surrounding sequences in the viral enhancer. However, the end of the insertion at nucleotide 142 was frequently preserved. The viral early and late promoter activity of the variant regulatory regions was tested in a luciferase reporter assay by using transfected NIH 3T3 cells. In relation to the standard-type DNA, all variants, including a G272T mutant, had much stronger late promoters. In contrast, the early promoter activity was influenced in a positive or negative direction by individual mutations. Also, the activity of the viral origin of DNA replication was affected by the sequence variation of the regulatory region, although the effects were smaller than for the late promoter. Analysis by Southern blotting and quantification using dot blots showed that approximately 10(3) copies of material related to the 220-bp insert in murine pneumotropic virus DNA was present in mouse and human DNA but not in Escherichia coli DNA. Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220-bp viral DNA segment. These data together with the nucleotide sequence analysis suggest that the 220-bp insertion is related to a transposable element of a novel type.

Animals↗

PubMed2006

Genetic analysis of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear localization of the origin recognition complex.

Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope-tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC-(2-5) or ORC-(1-5) complex; no other combinations of ORC subunits were detected. Moreover, only ORC-(1-5) was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC-(2-5) onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC-(2-5) in the nucleus. Because this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2.

Cell Line↗

PubMed2004

A novel intermediate in initiation complex assembly for fission yeast DNA replication.

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast.

Cell Cycle↗

PubMed1999

The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle.

The origin recognition complex (ORC) binds to the well defined origins of DNA replication in budding yeast. Homologous proteins in other eukaryotes have been identified but are less well characterised. We report here the characterisation of a fission yeast ORC complex (SpORC). Database searches identified a fission yeast Orc5 homologue. SpOrc5 is essential for cell viability and its deletion phenotype is identical to that of two previously identified ORC subunit homologues, SpOrc1 (Orp1/Cdc30) and SpOrc2 (Orp2). Co-immunoprecipitation experiments demonstrate that SpOrc1 forms a complex with SpOrc2 and SpOrc5 and gel filtration chromatography shows that SpOrc1 and SpOrc5 fractionate as high molecular mass complexes. SpORC subunits localise to the nucleus in a punctate distribution which persists throughout interphase and mitosis. We developed a chromatin isolation protocol and show that SpOrc1, 2 and 5 are associated with chromatin at all phases of the cell cycle. While the levels, nuclear localisation and chromatin association of SpORC remain constant through the cell cycle, one of its subunits, SpOrc2, is differentially modified. We show that SpOrc2 is a phosphoprotein which is hypermodified in mitosis and is rapidly converted to a faster migrating isoform as cells proceed into G(1) in preparation for S-phase.

Chromatin↗

PubMed1987

Fate of polyoma origin of replication after its direct introduction into mice.

Recently we have developed a method for direct introduction of calcium phosphate-precipitated DNA into newborn rats. To examine whether the foreign DNA can replicate, a plasmid containing a polyoma origin of replication was injected into newborn mice. The plasmid was found intact in liver and spleen and able to transform bacteria. The foreign DNA had disappeared by the seventh day after injection. Yet, the plasmid DNA containing the polyoma origin of replication had undergone replication in both the liver and the spleen.

Animals↗

PubMed1997

Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix.

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes.

Animals↗

PubMed1982

Tumorigenicity of human BK papovavirus plaque isolates, wild-type and plaque morphology mutant, in hamsters.

The polyoncogenic prototype human papovavirus BKV (Gardner's strain) produced clear, small and large plaques. Two small-plaque isolates (wt-500 and wt-502), as well as a large-plaque-forming isolate (wt-501), induced frequent brain tumors and occasional osteosarcomas (but no insulinomas) in hamsters. The large-plaque former (wt 501) was more tumorigenic than the two small-plaque mutant (pm-525), which had been rescued from hamster tumor cell lines and have small deletion near the origin of DNA replication, induced frequent brain tumors and insulinomas. Mutant 522 was about five times more tumorigenic than wt-501. These results support the hypothesis that the polyoncogenicity of prototype BKV is accounted for by the presence of BKV mutants.

Adenoma, Islet Cell↗

PubMed1992

Transformed rat pancreatic islet-cell lines established by BK virus infection in vitro.

Single-cell suspensions of primary rat pancreatic islet cells were infected with BK virus (BKV) prototype (Gardner) and the naturally occurring BKV strain (TU), isolated from human urine. These viral strains have different sequences in their non-coding control regions which contain promoter-enhancer elements and origin of DNA replication. Uninfected cell cultures disintegrated within 3 weeks, while a varying number of virus-infected cultures were immortalized and a majority exhibited focal areas of 2-dimensional growth. A significantly higher proportion of the BKV (TU)- than of the BKV (Gardner)-infected cultures were immortalized. Large tumor (T) antigen expression was evident in virus-infected cultures from day 4 post infection (p.i.), while insulin secretion decreased steadily during the first weeks of culture. Two cell lines were established from BKV (TU)-infected cultures. Line 5A4 was contact-inhibited, growing as a dense monolayer, while 6A3 demonstrated foci of 2-dimensional growth. Both cell lines have retained their morphology and T-antigen expression for approximately 130 passages. At high passages a low level of intracellular insulin, but no secretion into media, was detected. Based on standard biological tests (reduced serum requirements, growth at low cell density, anchorage-independent growth and tumor induction in nude mice) both cell lines have a fully transformed phenotype, but 6A3 appears to be more malignantly transformed. Since both cell lines were established simultaneously from the same primary cells with the same virus batch, they provide an opportunity to study the transforming mechanisms of BKV in a relative context.

Animals↗

PubMed1994

DNA of lymphoma-associated herpesvirus (HVMF1) in SIV-infected monkeys (Macaca fascicularis) shows homologies to EBNA-1, -2 and -5 genes.

We have characterized a new Epstein-Barr-virus(EBV)-like herpesvirus associated with lymphomas of SIV-infected cynomolgus (Macaca fascicularis) monkeys and propose that this virus is designated herpesvirus macaca fascicularis I (HVMFI). Genomic regions in HVMF1 of potential significance for tumor pathogenesis were analyzed by Southern blotting, PCR and sequencing, and compared with human EBV DNA. Virus from 7 SIV-associated lymphomas and one lymphoma-derived cell line were shown to share homology with the EBNA1- and EBNA2-coding regions of EBV, while some homology to EBV-LMP1 was detectable only at low-stringency hybridization. Homologous regions to the long internal repeat (IR1; BamHI W), the EBER1 and 2 and the latent origin of DNA replication (oriP) could also be demonstrated in HVMF1. These coding regions, except IR1, showed restriction-enzyme maps different from those of EBV. Sequencing of the EBNA5 homologous region of HVMF1 DNA, corresponding to exons W1 and W2, showed 65% homology to the EBV exons W1 and W2, and 80% to the whole region including the intron. Since EBNA5 has been reported to bind tumor-suppressor proteins p53 and Rb in vitro, the HVMF1 homology could be important for the lymphomagenic capacity of this monkey herpesvirus.

Amino Acid Sequence↗

PubMed1986

Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions.

We have examined the relative sensitivities of different genetic functions to damage induced by u.v. light. For this purpose we have introduced defined amounts of damage into specific regions of the genome of SV40 and have determined the effect of the damage on the survival of transfected viral DNA. We found that the lethal effect of the damage depends on its location within the viral genome. The region most sensitive to damage contains the transcriptional promotors and enhancers for the early and late viral genes plus part of the origin of DNA replication. Lesions within this regulatory region are 3.2-fold more effective in inactivating viral DNA than is the same amount of damage randomly distributed throughout the viral genome. The region least sensitive to damage lies within the coding portion of the viral coat protein genes, which are expressed only late in infection and would therefore be transcribed from undamaged progeny viral genomes, provided DNA replication occurs. Damage within this region is only 45% as effective in inactivating viral DNA as are randomly distributed lesions. Thus there is a 7-fold difference in the lethal effect of DNA damage within the most sensitive and least sensitive regions of the viral genome. Intermediate sensitivities are observed within the transcribed portion of the viral A gene, coding for the T antigen whose expression is required early in infection, and in a region at the terminus of DNA replication. The sum of the individual sensitivities for all regions of the SV40 genome is equal to the total sensitivity of viral DNA subjected to random damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1976

SV40 DNA sequences as an example of the structure of genes functioning in animal cell nuclei.

Recent studies of the structure of messenger RNA have demonstrated the existence of untranslated sequences of the 3' and 5' end of the messages. In addition analysis of transcription in vitro has indicated that the nucleotide sequence U6 purine may be part of a transcription termination signal in prokaryotes. Recently it has been possible to determine the sequence of extensive portions of the DNA of SV40 virus. This article reviews the analogies between certain of these sequences and sequences available from prokaryotic messengers and DNAs. Unusual structures, including blocks of AT-rich and GC-rich segment sections and symmetric regions in the DNA near the origin of DNA replication, have been demonstrated and the distribution of stretches of 6 or more deoxyadenylic acids in the DNA of SV40 is consistent with some rho for these sequences in animal cells, either as terminators of transcription or as sites where degradation of transcripts is initiated or sites related to the selective rejection or degradation of transcipts.

Animals↗

PubMed1992

Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.

A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.

Animals↗

PubMed1998

Expression systems for study of mycobacterial gene regulation and development of recombinant BCG vaccines.

Successful genetic engineering of mycobacteria is crucial for developing new approaches to combat tuberculosis as well as for dissecting out the molecular basis of pathogenesis of Mycobacterium tuberculosis. We have constructed a Mycobacterium-Escherichia coli shuttle expression vector pSD5. It carries a modular expression cassette which provides sites for cloning of promoters, a ribosome binding site (RBS) with an appropriately placed initiation codon and multiple cloning sites for cloning the genes of interest. We also constructed pDK20, an integration proficient derivative of pSD5, by incorporating mycobacteriophage L5 integration signals in lieu of the origin of DNA replication for mycobacteria. This vector permits stable expression of genes in M.bovis BCG, M.smegmatis, and M.tuberculosis under the transcriptional control of a mycobacterial promoter. These vectors enable the expression of a gene to be regulated by several hundred fold depending upon the strength of mycobacterial promoter. We propose that expression of protective antigens using an appropriate promoter derivative of pDK20 should help in development of recombinant BCG vaccines that can induce an optimal immune response from the host. We have further employed the integration proficient expression system for comparing the efficiency and specificity of transcriptional recognition in M.bovis BCG, M.tuberculosis, and M.smegmatis. We show that fast growing M.smegmatis and slow growing M.tuberculosis and M.bovis BCG recognize mycobacterial promoters with comparable efficiency inspite of differences in their growth rates.

BCG Vaccine↗

PubMed2002

DNA plasmid transmission in yeast is associated with specific sub-nuclear localisation during cell division.

Circular plasmids in yeast carrying only an origin of DNA replication (ARS) exhibit maternal inheritance bias (MIB) and are poorly transmitted from mother to daughter cell during division. A variety of different sequences that overcome MIB have been described, including centromeric sequences (CEN), telomere-associated repeats, silencer sequences and a specific system encoded by the endogenous 2 micron circle plasmid requiring the cis-acting locus STB and the proteins Rep1 and Rep2. In each case, DNA segregation between mother and daughter cells is dependent on DNA-protein interactions. Using plasmids carrying multiple copies of a lac repressor binding sequence, we have localised DNA molecules in the yeast nucleus using a green fluorescent protein (GFP)-lac repressor fusion protein. We compared GFP localised plasmids carrying a centromere sequence with plasmids based on 2 micron circle carrying or lacking the STB sequences required for their segregation. We show that GFP localised plasmid carrying the complete STB locus co-localises with the plasmid proteins Rep1 and Rep2 to discrete chromatin sites. These sites are distinct from both the telomeres and from sites of cohesin binding. Deletion of the region of STB essential for the stability of the plasmid, leads to a loss of plasmid association with chromatin, relocalisation of plasmids towards the nuclear periphery, and a decrease in the Rep1 protein associated with the plasmid. We conclude that specific plasmid localisation is likely to be important in the overcoming of MIB in yeast.

Cell Division↗

PubMed1995

Repression of gene expression at the beginning of mouse development.

The transition from maternal to zygotic gene expression in the mouse occurs in the 2-cell embryo. Previous studies in which DNA was injected into 2-cell embryos revealed that transcription promoters and origins of DNA replication are strongly repressed in cleavage stage embryos unless linked to an embryo-responsive enhancer. Repression also occurs when DNA is injected into the paternal pronucleus of a 1-cell embryo and the injected embryo subsequently undergoes mitosis, except that repression is no longer relieved by enhancers. Here we extend this observation to maternal pronuclei in 1-cell embryos and show that this repression could not be relieved either by linking the promoter to an embryo-responsive enhancer or by inducing hyperacetylation of chromatin inorder to increase its accessibility to transcription factors. However, repression could be relieved by transplanting the injected pronucleus to a 2-cell embryo, even when the recipient cell subsequently underwent mitosis. Both the extent of promoter repression and the ability of enhancers to stimulate promoter activity increased as development proceeded from the early 2-cell stage to the 4-cell stage. Once repression was established in late 2-cell embryos, transplanting an injected 2-cell embryo nucleus back to an early 1-cell embryo failed to restore activity to the injected promoter, even when it was linked to an enhancer. These and other data demonstrate that cytoplasmic factors appear during formation of a 2-cell embryo that can repress promoter activity and activate enhancer activity. These factors are absent from the paternal pronucleus and cytoplasm of early (S-phase arrested) 1-cell embryos. Moreover, the cytoplasm of early 1-cell embryos appears to lack the ability to reprogram expression of genes once they have progressed to the late 2-cell stage in mouse development.

Animals↗

PubMed2001

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein.

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.

Alleles↗

PubMed2001

Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.

Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

Animals↗