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PubMed1976

The effect of 313 nanometer light on initiation of replicons in mammalian cell DNA containing bromodeoxyuridine.

After unifilar substitution of thymine with bromouracil in either human HeLa or bovine cells, exposure to 313 nm light inhibits initiation of clusters of replicons. Since this treatment results in damage only to DNA and because the effect is the same as observed after 100-1000 rads of X-ray irradiation to the same cells, we infer that the effect of the later treatment is also mediated almost exclusively by DNA damage.

Bromodeoxyuridine↗

PubMed1982

Transposon Tn3-mediated replicon fusion.

To investigate inter-replicon transposition of Tn3, we used the cosmid-phage lambda packaging system coupled with density gradient fractionation and isolated recombinant molecules of different sizes. Cosmids derived from ampicillin-resistance-transducing phage were classified into four groups: (1) cosmid-Tn3 donor cointegrates considered as Tn3 transposition intermediates, (2) similar cointegrates carrying deletions of one copy of Tn3 and of adjacent cosmid DNA sequences, (3) cosmids carrying a single Tn3 insertion, and (4) cosmids carrying two independent Tn3 insertions. Genetic and biochemical studies indicated that cosmids isolated from ampicillin-resistance transductants were derived from the authentic cosmid-Tn3 donor cointegrate intermediates.

Bacteriophage lambda↗

PubMed1982

Site-specific recA-independent recombination (fusion) of pMB8-related replicons in Escherichia coli in the region of the replication origins.

Escherichia coli recA+ and recA- cells were co-transformed with a mixture of pMB9 (Tcr) and pST8-26 (Apr, pBR325 derivative) plasmid DNAs followed by selection on plates containing both tetracycline and ampicillin. A set of stable Tcr Apr derivatives was isolated from these transformants. Many of the stable Tcr Apr segregants contained fused pMB9::pST8-26 plasmids with lengths that were about 0.8 kb longer than the sum of the lengths of the parental plasmids; one plasmid (pTF8) was about 1.5 kb shorter. The fusion was not stimulated by UV irradiation of co-transformants and occurred both in recA+ and recA- genetic backgrounds. Restriction analysis of the fused plasmids showed the two replicons were in the same relative orientation, and also indicated unique points of fusion in most cases (in 9 out of 10) which are localised within the 1.6-kb regions around the replication origins (RO). Because the fusion of plasmids of the type used in this study was not described before we have tentatively named it RO-fusion (Replication-Origin-fusion).

Chromosome Mapping↗

PubMed1968

Replicating units (replicons) of DNA in cultured mammalian cells.

Exponentially growing L5178Y mouse leukemic cells were incubated in the presence of 5'-bromodeoxyuridine (BUdR) for about 4 hr, transferred to the nonBUdR-containing medium for a certain period (t hours), and then pulse-labeled with TdR-(3)H for 10 min. When DNA isolated from these cells was subjected to CsCl gradient centrifugation, the (3)H-activity was found to shift gradually from the heavy BUdR-containing peak to the light nonBUdR-containing peak with increasing time t. The average time required for the complete shift of (3)H-activity from the heavy to the light DNA fraction was 2.76 hr. Taking this as the average replicating time and the size of DNA fragments in the present preparation as 1.3 x 10(7) daltons, the rate of replication was found to be 2.1 nucleotides per strand per replicon per sec. By taking the upper limit of the average replicating time as the S period (7.3 hr), various characteristics of the replicating units, such as the lower and upper limits of average size, the average replicating time, the average number of replicating units, etc., were calculated (see Table I).

Animals↗

PubMed1977

Inhibition of initiation of HeLa cell replicons by methyl methanesulfonate.

Methyl methanesulfonate (MMS) in the culture medium inhibits the rate of DNA synthesis in HeLa cells in a dose-dependent manner. By using short (5 min) incubations with [3H]thymidine and analyzing the newly made DNA by velocity sedimentation on alkaline sucrose gradients, we found that the first affect of MMS on DNA replication, at 0.5 h after treatment, was inhibition of initiation of replicons. Recovery from this effect seemed to have begun by 2 3/4 h after treatment. The second effect of MMS, which was evident at 2 h after treatment, was to slow or block chain elongation.

Centrifugation, Density Gradient↗

PubMed2002

Evaluation of FIV protein-expressing VEE-replicon vaccine vectors in cats.

Venezuelan equine encephalitis (VEE) virus-replicon particles (VRP) were used to generate feline immunodeficiency virus (FIV) Gag- and ENV-expressing vaccine vectors. Serum and mucosal FIV-specific antibody was detected in cats immunized subcutaneously, once monthly for 5 months, with FIV-expressing VRP. Expansion of the CD8+ L-selectin negative phenotype and transient CD8+ noncytolytic suppressor activity were seen in cats immunized with FIV-expressing or control VRP. Despite induction of FIV-specific immune responses and nonspecific suppressor responses, all cats became infected following vaginal challenge with high dose, pathogenic cell-associated FIV-NCSU(1) although relative early maintenance of CD4+ cells was seen in FIV-immunized cats.

Animals↗

PubMed1998

pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli.

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.

Chromosomes, Bacterial↗

PubMed2004

The replicon revisited: an old model learns new tricks in metazoan chromosomes.

The origins of DNA replication were proposed in the replicon model to be specified genetically by replicator elements that coordinate the initiation of DNA synthesis with gene expression and cell growth. Recent studies have identified DNA sequences in mammalian cells that fulfil the genetic criteria for replicators and are beginning to uncover the sequence requirements for the initiation of DNA replication. Mammalian replicators are com- posed of non-redundant modules that cooperate to direct initiation to specific chromosomal sites. Conversely, replicators do not show strong sequence similarity, and their ability to initiate replication depends on the chromosomal context and epigenetic factors, as well as their primary sequence. Here, we review the properties of metazoan replicators, and discuss the genetic and epigenetic factors that determine where and when DNA replication is initiated.

Animals↗

PubMed2001

RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon.

The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon. RepC is essential for replication, and RepA and RepB play a role in plasmid segregation. It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number. RepA is also a trans-incompatibility factor. To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site-directed mutagenesis of the putative -35 and -10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon.

Bacterial Proteins↗

PubMed2004

In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium.

We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on in planta assembly of functional viral vectors from separate pro-vector modules. With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver various modules that are assembled inside the cell with the help of a site-specific recombinase. The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating a fully functional RNA replicon. The proposed protocol allows us, by simply treating a plant with a mixture of two or more agrobacteria carrying specific prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform the various engineering steps normally required with alternative protocols. The process described here is very fast (expression requires 3-4 days); it provides very high protein yield (up to 80% of total soluble protein); more than before, it is carried out using in vivo manipulations; it is based on prefabricated genetic modules that can be developed/upgraded independently; and it is inherently scalable.

DNA, Bacterial↗

PubMed2005

Modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles.

Measles remains a major worldwide problem partly because of difficulties with vaccination of young infants. New vaccine strategies need to be safe and to provide sustained protective immunity. We have developed Sindbis virus replicon particles that express the measles virus (MV) hemagglutinin (SIN-H) or fusion (SIN-F) proteins. In mice, SIN-H induced high-titered, dose-dependent, MV-neutralizing antibody after a single vaccination. SIN-F, or SIN-H and SIN-F combined, induced somewhat lower responses. To assess protective efficacy, juvenile macaques were vaccinated with a single dose of 10(6) or 10(8) SIN-H particles and infant macaques with two doses of 10(8) particles. A dose of 10(8) particles induced sustained levels of high-titered, MV-neutralizing antibody and IFN-gamma-producing memory T cells, and most monkeys were protected from rash when challenged with wild-type MV 18 months later. After challenge, there was a biphasic appearance of H- and F-specific IFN-gamma-secreting CD4+ and CD8+ T cells in vaccinated monkeys, with peaks approximately 1 and 3-4 months after challenge. Viremia was cleared within 14 days, but MV RNA was detectable for 4-5 months. These studies suggest that complete clearance of MV after infection is a prolonged, phased, and complex process influenced by prior vaccination.

Aging↗

PubMed1979

Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin.

The circular genome of simian virus 40 is a model mammalian replicon, containing a unique origin of replication (ori) and coding for a protein (SV40 T antigen) known to be involved in initiation of viral DNA replication and to bind in vitro to the origin region. Mutations within the ori sequence lead to defective viral DNA replication and the formation of small viral plaques after infection of a cell monolayer. Second-site revertants (pseudorevertants) of ori mutants were isolated by random local mutagenesis of mutant DNA followed by transfection of cultured cells and the selection of large plaques. In each case, reversion of the plaque phenotype was associated with an increased rate of viral DNA replication. The second-site mutations that suppressed the replication defects were localized by in vitro recombination or marker rescue experiments to the gene for T antigen. Their map positions differ from those of previously described T antigen mutants, possibly reflecting a specific ori-binding domain of T antigen. From these results we infer that T antigen interacts with the ori signal during virus development as it does in vitro and that this interaction regulates the rate of viral DNA replication.

Antigens, Neoplasm↗

PubMed1984

In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon.

A mini-Mu bacteriophage containing a high copy number plasmid replicon was constructed to clone genes in vivo. A chloramphenicol resistance gene for independent selection and the lacZYA operon to form gene fusions were also incorporated into this phage. This mini-Mu element can transpose at a high frequency when derepressed, and it can be complemented by a helper Mu prophage for lytic growth. DNA sequences that are flanked by two copies of this mini-Mu can be packaged along with them. After infection, homologous recombination can occur between the mini-Mu sequences, resulting in the formation of plasmids carrying the transduced sequences. lac operon fusions can be formed with promoters and translation initiation sites on the cloned sequences in the resulting plasmids. The utility of this system was demonstrated by cloning genes from eight different Escherichia coli operons and by identifying lac fusions to the regulated araBAD operon among clones selected for the nearby leu operon.

Cloning, Molecular↗

PubMed1995

Granulocyte macrophage-colony stimulating factor-dependent replication of polyoma virus replicon in hematopoietic cells. Analyses of receptor signals for replication and transcription.

Granulocyte macrophage-colony stimulating factor (GM-CSF) stimulates proliferation of various hematopoietic cells. Using cytoplasmic deletion mutants of the human GM-CSF receptor (hGMR) beta subunit and tyrosine kinase inhibitors, we previously showed that distinct signaling pathways of hGMR are involved in the induction of c-fos/c-jun mRNAs and of c-myc mRNA/cell proliferation. We used polyoma virus (Py) replicon to analyze the initiation of DNA replication induced by hGM-CSF in mouse BA/F3 pro-B cells expressing hGMR. hGM-CSF efficiently stimulated Py replication in the presence of Py enhancer and Py large T antigen supplied in trans. Analyses of Py enhancer mutants revealed that hGM-CSF promoted Py replication and activated transcription of the Py early promoter through the PEA3/PEBP5 region of Py enhancer. The membrane proximal region of hGMR beta subunit is required for activation of PEA3/PEBP5-dependent replication which is also required for activation of DNA synthesis in the host cells. In contrast, a more distal region which is essential for activation of c-fos and c-jun genes is required for the PEA3/PEBP5-dependent transcription of Py early promoter. These results indicate that distinct signaling pathways of hGMR are required to activate PEA3/PEBP5-dependent replication and transcription although the same enhancer is required for both activities.

Animals↗

PubMed1982

Neoplastic transformation of Syrian hamster embryo cells by bisulfite is accompanied with a decrease in the number of functioning replicons.

Bisulfite, a chemical that at neutral pH does no induce mutations at two loci in Chinese hamster V-79 cells, induces transformation of Syrian hamster embryo cells. Although bisulfite affects DNA metabolism, there was no evidence of bisulfite-induced DNA damage. Bisulfite induced no excision repair replication, caused no DNA strand breaks detectable in alkaline sucrose gradients, had no effect on the size distribution of DNA nascent daughter strands, and did not affect excision on post-replication repair of u.v.-induced damage. However, bisulfite did induce a dose-dependent decrease in the rate of DNA replication per cell, apparently due to a reduction in the number of functioning replicons. Since the data indicate that bisulfite causes no detectable DNA damage, it appears that bisulfite induces transformation by a non-mutagenic event that could involve the inhibition of semi-conservative DNA synthesis.

Animals↗

PubMed1995

Tandem repeats of the 5' non-transcribed spacer of Tetrahymena rDNA function as high copy number autonomous replicons in the macronucleus but do not prevent rRNA gene dosage regulation.

The rRNA genes in the somatic macronucleus of Tetrahymena thermophila are normally on 21 kb linear palindromic molecules (rDNA). We examined the effect on rRNA gene dosage of transforming T.thermophila macronuclei with plasmid constructs containing a pair of tandemly repeated rDNA replication origin regions unlinked to the rRNA gene. A significant proportion of the plasmid sequences were maintained as high copy circular molecules, eventually consisting solely of tandem arrays of origin regions. As reported previously for cells transformed by a construct in which the same tandem rDNA origins were linked to the rRNA gene [Yu, G.-L. and Blackburn, E. H. (1990) Mol. Cell. Biol., 10, 2070-2080], origin sequences recombined to form linear molecules bearing several tandem repeats of the origin region, as well as rRNA genes. The total number of rDNA origin sequences eventually exceeded rRNA gene copies by approximately 20- to 40-fold and the number of circular replicons carrying only rDNA origin sequences exceeded rRNA gene copies by 2- to 3-fold. However, the rRNA gene dosage was unchanged. Hence, simply monitoring the total number of rDNA origin regions is not sufficient to regulate rRNA gene copy number.

Animals↗

PubMed2002

Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins.

Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.

Animals↗

PubMed1996

In vitro-generated 'inverse' chimeric Coleus blumei viroids evolve in vivo into infectious RNA replicons.

The three Coleus blumei viroids (CbVd) CbVd 1-RL, CbVd 2-RL and CbVd 3-RL occur naturally in plants of Coleus blumei cultivar (cv.) 'Ruhm von Luxemburg'. CbVd 2-RL is a viroid chimera made up of the right half of CbVd 1-RL and the left half of CbVd 3-RL. Using the cDNAs of the left half of CbVd 1-RL and the right half of CbVd 3-RL, 'inverse' viroid chimeras (CbVd-Ch1 and CbVd-Ch2) were constructed in vitro as dimeric cDNA units. The in vitro-generated cDNA-transcripts of CbVd-Ch1 and CbVd-Ch2 were used to inoculate plants of Coleus blumei cv. 'Ruhm von Luxemburg' harbouring CbVd 1-RL and CbVd 3-RL, leading to the emergence of new viroid-like RNA replicons, named CbVd A-1 and CbVd A-2, that hitherto have not been found in Coleus plants. The sequences of CbVd A-1 and CbVd A-2 are not identical to the corresponding sequences of the cDNA-transcripts of CbVd-Ch1 and CbVd-Ch2, but show a deletion of a segment at the border of the two original halves as well as two U to A transitions. When the cDNA-transcripts of CbVd-Ch1 and CbVd-Ch2 were used to inoculate plants of Coleus blumei cv. 'Scarlet Dragonfly' that were either viroid-free or infected with CbVd 1-RL and CbVd 3-RL, no new viroid-like RNAs emerged. However, the cDNA-transcripts of CbVd A-1 and CbVd A-2 are infectious by mechanical inoculation of viroid-free plants of cv. 'Scarlet Dragonfly', thus proving their viroid nature.

Base Sequence↗