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PubMed1998

Remodeling of chromatin loops does not account for specification of replication origins during Xenopus development.

We have investigated the possible relationship between replicons and chromatin loops during Xenopus development. In early embryos, replication of the ribosomal RNA genes (rDNA) can initiate at apparently any sequence. Nevertheless, the need for a regular spacing of replication origins suggests that some periodic chromatin folding might dictate which sites are actually used for initiation. After the midblastula transition, replication initiation is restricted to the rDNA intergenic spacers. A remodeling of chromatin folding could account for this change in origin usage. Here, it is reported that nuclear matrix anchorage of the Xenopus rDNA occurs at multiple, apparently random sequences, throughout embryonic development as well as in adult cells. In vitro matrix rebinding assays confirmed the lack of specific anchoring sequences in the rDNA, before as well as after specific replication origins are established. Thus, no change in loop attachment sites could explain the change in origin usage at this locus. Nonspecific loop anchorage was a special feature of the rDNA locus, since the same nuclear matrices were able selectively to bind the scaffold attachment region (SAR) of the Drosophila histone gene cluster in vitro. Blastula and gastrula nuclear matrices bound a higher amount of SAR sequences than matrices from later stages or adult cells. This developmental change in SAR binding might explain the increase in size of the bulk of genomic DNA loops that occurs after the gastrula stage. However, no change in chromatin loop organization that could explain the midblastula stage transition from small to large replicons was observed.

Animals↗

PubMed1999

Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors.

Virion sense gene replacement derivatives of maize streak virus (MSV) were constructed with selectable marker expression cassettes based on the bialaphos resistance gene (bar) and the CaMV 35S promoter. The effect on replication of increasing the genomic size was tested by including: (1) the 550-bp maize adhI intron and 68-bp TMV omega RNA leader sequences upstream of the bar genes; and (2) a fusion between the bar and E. coli glutathione reductase (gor) genes. Three recombinant viral vectors ranging in size from 2.7 kb to 4.8 kb replicated efficiently in biolistically transfected cells of suspension cultured Black Mexican sweetcorn (BMS) cells. Deletions greater than 39 bp 3' of the stemloop sequence in the LIR adversely affected replicon release. Transformed bialaphos-resistant BMS cell lines were generated with all three vectors containing the bar gene: between 38 and 60% of cell lines contained replicating viral episomes. The replicons were structurally stable, replicated to copy numbers of over 500 per haploid genome, and were detected for more than one year after introduction. We noted significant enhancement of bar gene expression, both at the protein and RNA levels, associated with the presence of episomal vector DNA. The maize adhI intron and TMV omega RNA leader sequences did not seem to have a significant effect on bar gene expression from replicating constructs, although expression from controls was enhanced. The results suggest that MSV-based constructs would provide a useful system for long-term gene amplification in cereal cell culture systems.

Acetyltransferases↗

PubMed1979

Dependence of mammalian DNA replication on DNA supercoiling. II. Effects of novobiocin on DNA synthesis in Chinese hamster ovary cells.

Novobiocin, an inhibitor of gyrase-induced DNA supercoiling and DNA replication in prokaryotes, inhibited the incorporation of DNA precursors into DNA in both intact and permeable Chinese hamster ovary cells; much higher concentrations were required for permeable cells, in which no new replicons were initiated. Nucleoids were prepared from cells that were incubated for 60 min with 200 micrograms/ml novobiocin, made permeable, and incubated with 0--50 micrograms/ml ethidium bromide. Sedimentation of the nucleoids in neutral sucrose gradients suggested that the number of supercoils in the average nucleoid had been reduced by prior incubation with novobiocin. In intact cells, novobiocin is required inside the cell for continued inhibition of DNA synthesis, suggesting that it does not act directly on the DNA. Alkaline sucrose gradient profiles of DNA synthesized in the presence of novobiocin in intact cells indicated that the drug inhibited replicon initiation while having little if any effect on chain elongation. These data are consistent with the idea that an activity similar to the bacterial gyrase generates supercoils in mammalian DNA and produces the proper conformation for the initiation of DNA replication.

Animals↗

PubMed1983

Two discrete DNA replication intermediates are formed in melphalan-treated cells.

In undisturbed cells one can detect a discrete population of 10 kb DNA replication intermediates, which indicates the existence of gaps spaced about 10 kb away from each other in the newly synthesized DNA. In melphalan-treated cells one can detect both 10 kb and 20 kb DNA intermediates, indicating that in such cells the gaps present in a replicon are not filled at the same time which allows the detection of a molecule which is formed by the joining of two 10 kb DNA intermediates. In contrast in undisturbed cells it is likely that the filling of the gaps present in a replicon occurs at the same time which prevents the detection of a 20 kb DNA intermediate.

Cell Line↗

PubMed1985

Inhibition of initiation of simian virus 40 chromosome synthesis by dihydroxyanthraquinone.

The mode of action of dihydroxyanthraquinone, a new antitumor drug, on eucaryotic chromosome structure and function was investigated using simian virus 40 as a model system. Dihydroxyanthraquinone specifically inhibited initiation of viral replicons. Little or no viral DNA synthesis was recovered in cells after the removal of the drug. Elongation and termination of DNA already initiated could proceed continuously to completion in drug-treated cells. The drug appeared to be stably associated with viral chromosomes in cells. The irreversible inhibition of replicon initiation might contribute to its anti-proliferative and anti-neoplastic activity.

Animals↗

PubMed1990

Physical relationship between a gene and its origin of replication in Physarum polycephalum.

Taking advantage of the natural synchrony of the S-phase within the plasmodium of Physarum polycephalum, we extracted highly synchronous DNA samples at precise time points in early S-phase. We then separated, by electrophoresis under denaturating conditions, the newly synthesized DNA strands of the nascent chromosomal replicons from the parental DNA template. Using the cDNA clone of the early-replicating LAV1-2 gene as a probe, we could establish by filter hybridization that the elongation rate of the replicon which encompasses this gene is constant, at a rate of 1 kb/min during the first 30 min of S-phase. The smallest replication intermediate (RI) that we have detected by probing with the LAV1-2 cDNA was 5 kb long, suggesting that the LAV1-2 gene and its origin of replication are closely associated within the chromosome. This procedure should facilitate the mapping of replication origins within the genome of Physarum.

DNA Probes↗

PubMed1980

A replication model for sister-chromatid exchange.

A model for the production of sister-chromatid exchanges is presented, based on the idea that double-strand breaks are generated at junctions between a completely duplicated replicon cluster and a partially duplicated replicon cluster. Agents that induce absolute blocks to DNA fork displacement will cause this condition to persist longer than normal, whereas agents that inhibit initiation of whole clusters will rarely cause it at all. During the blunt-end repair of the double-strand breaks, sister-chromatod exchange would be initiated when daughter strands of a duplicated cluster recombine with the parental strands of the partially replicated cluster. When the latter finishes replication, sister-chromatid exchange would be completed.

Crossing Over, Genetic↗

PubMed1981

Radioresistant DNA synthesis: an intrinsic feature of ataxia telangiectasia.

DNA synthesis in 6 ataxia telangiectasia (AT) cell strains was much more resistant to X-irradiation than was DNA synthesis in normal human diploid cells. 3 of the cell strains tested have been classified as proficient in repair replication. These data, along with those reported elsewhere, strongly suggest that radioresistant DNA synthesis is an intrinsic feature of this disease. The radioresistance of DNA synthesis in AT cells is primarily due to a reduced inhibition of replicon initiation compared to that occurring in normal cells, but DNA chain elongation is also more radioresistant in AT cells. The small inhibition of DNA synthesis that does occur in AT cells at doses up to 2000 rad is almost exclusively due to inhibition of replicon initiation, and not to inhibition of chain elongation, as would be expected from results with normal human cells or from previous studies with established cell lines.

Ataxia Telangiectasia↗

PubMed1982

The effects of bifunctional alkylating agents on DNA synthesis in sensitive and resistant Yoshida cells.

The effects of the alkylating agents methylene dimethanesulphonate (MDMS), sulphur mustard (SM) and methyl methanesulphonate (MMS) on DNA synthesis have been investigated in Yoshida sarcoma cell lines sensitive (YS) and resistant (YR) to these agents. Measurement of overall DNA synthesis by [3H]thymidine uptake into DNA indicated that both MDMS and SM, but not MMS, induced a greater depression of DNA synthesis in YS cells than in YR cells. Analysis of the sizes of newly synthesised strands of DNA by pulse labelling and alkaline sucrose gradient sedimentation revealed that MDMS inhibited the initation of new replicons and also reduced the size of new DNA strands. The relative extents of inhibition of replicon initation in MDMS-treated YS and YR cells were similar and reduced by caffeine while YR cells were comparatively less sensitive to the effect of MDMS on the sizes of DNA molecules synthesised. Similarly, YR cells apparently synthesised longer DNA strands following SM treatment compared to YS cells suggesting an association between the size of DNA molecules synthesized and the sensitivity of Yoshida cells to bifunctional alkylating agents.

Alkylating Agents↗

PubMed1978

Size distribution and maturation of newly replicated DNA through the S and G2 phases of Physarum polycephalum.

The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as "Okazaki" fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4-5 X 10(5) daltons per min at 26 degrees C, corresponding to the joining of 6-8 Okazaki fragments. The resulting 35-45S (1.1-2.2 X 10(7) daltons) DNA molecules may correspond to the Physarum "replicon." The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation.

Cell Cycle↗

PubMed1984

Structural and functional analysis of the par region of the pSC 10 1 plasmid.

Three distinct segments (the partition-related, or PR segments) within the 370 bp par region of pSC101 have been shown by deletion analysis to be involved in partitioning of the plasmid to daughter cells. The two lateral segments are direct repeats, each of which potentially can pair with an inverted repeat located between them to form a hairpin-loop structure. Deletion of either lateral segment, together with the middle segment, results in plasmid instability (the Par- phenotype). Deletion of one PR segment yields a stable plasmid that nevertheless shows reduced ability to compete with a coexisting wild-type derivative of the same replicon (the Cmp- phenotype). Deletion of all three segments results in a rate of plasmid loss far in excess of that predicted from the observed copy number of the plasmid. Analysis of the segregation properties of these mutants and of temperature-sensitive and high copy number derivatives of the pSC101 replicon suggests a model in which the par function allows the nonreplicating plasmids of the intracellular pool to be counted as individual molecules, and to be distributed evenly to daughter cells. In the absence of par, the multicopy pool of plasmids behaves as a single segregation unit.

Base Composition↗

PubMed1988

Comparative analysis of five related Staphylococcal plasmids.

The genomic organization of five small multicopy staphylococcal plasmids comprising the pT181 family has been analyzed. In addition to pT181, the family presently includes the streptomycin resistance plasmid pS194 and the chloramphenicol resistance plasmids pC221, pC223, and pUB112. Although they belong to five different incompatibility groups, the five plasmids have similar basic replicons, use the same basic copy control mechanism, and have a common structural organization. It has been demonstrated previously that pT181 and pC221 encode trans-active replication proteins (RepC and RepD, respectively) which specifically recognize the respective plasmid's origin of replication in both cases is initiated by site-specific nicking and 3' extension. The other three plasmids in this family encode similar replication proteins; 63% of the predicted amino acid residues are identical for all five and the least similar pair shows 75% identity at the amino acid level. However, despite this homology, the replication proteins and origins of replication of different members in this family did not show cross complementation in vivo. Outside of the basic replicon, which comprises about one-third of each plasmid's genome, functional organization is also conserved. The resistance determinants are all located in the same position, immediately downstream of the replication protein coding sequence, and all are transcribed in the same direction. The three chloramphenicol resistance determinants encode highly homologous chloramphenicol transacetylases which are unrelated to the tet and str gene products. Three of the five plasmids form relaxation complexes and the involved genome segments are closely related. The other two are not homologous to these three in the corresponding region, but are homologous to each other and encode a site-specific recombinase, Pre. It is suggested that the replication, resistance, and relaxation complex regions of these plasmids can be regarded as conserved segments ("cassettes") assembled in various combinations, but always with the same spatial arrangement.

Amino Acid Sequence↗

PubMed1988

A single amino acid difference between Rep proteins of P1 and P7 affects plasmid copy number.

P1 and P7 are closely related plasmid prophages which are members of the same incompatibility group. We report the complete DNA sequence of the replication region of P7 and compare it to that of P1. The sequence predicts a single amino acid difference between the RepA proteins of these two plasmids, no differences in methylation sites or regions where dnaA protein is expected to bind, and no difference in the spacing of the major features of the two replicons. A P1 replicon with a mutation in repA, the gene that encodes an essential replication protein, is complemented for replication by providing either the P1 RepA protein (RepA1) or the P7 RepA protein (RepA7) in trans. Furthermore, when either of these proteins is supplied in trans, the plasmid copy number of P1 cop mutants drops to that of P1 cop+. However, when RepA7 is supplied, the copy number of P1 cop and P1 cop+ is higher than that when RepA1 is supplied. This indicates that the single amino acid difference between the two versions of the RepA protein plays an important role in determining the plasmid copy number.

Amino Acids↗

PubMed1986

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation.

The rate of DNA synthesis after gamma-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of gamma-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.

Cell Cycle↗

PubMed1993

Analysis of mutagenesis induced by expression of retroviral reverse transcriptase in Escherichia coli.

We showed that expression of reverse transcriptase from HIV or MuLV resulted in exceptionally high levels of mutagenesis in E coli. We observed high rates of mutagenesis in plasmid genes when reverse transcriptase was expressed off that plasmid. Although very high rates were observed in cis, our experiments could not detect mutagenic events in markers in other replicons (ie in trans). These results suggest mutagenic events occur preferentially on the same replicon in which the reverse transcriptase is encoded.

Escherichia coli↗

PubMed1990

A bifunctional Streptomyces-E. coli promoter-probe vector.

A bifunctional Streptomyces-E. coli promoter probe vector, pULJA30, has been developed to isolate and characterize nucleotide sequences involved in transcription initiation and regulation. The vector is derived from plasmid pIJ486, carries the pIJ101 replicon and utilizes the promoterless aminoglycoside phosphotransferase (neo) as indicator gene. Important features of the new vector include: wide Streptomyces host range and as high a plasmid copy number as the parental pIJ486, an upstream transcriptional terminator (toop) and a polylinker sequence with unique sites for BamHI and BglII for flexible cloning, fragment re-isolation and direct sequencing of promoter-active inserts. pULJA30 also has an E. coli replicon (from pBR322) and the possibility of selection in Streptomyces and E. coli by using the tsr, neo and bla genes, which makes it very convenient to test the comparative functionality of Streptomyces promoters in E. coli.

Base Sequence↗

PubMed1992

Plasmid maintenance in Bacillus stearothermophilus is strain-dependent.

We studied the segregational stability of plasmids based on pTB913, a 4.5-kb rolling-circle plasmid derived from the thermophilic Bacillus plasmid pTB19. In Bacillus stearothermophilus the stability of pTB913 derivatives appeared to be strain-dependent. In strain CU21 large amounts of single-stranded pTB913 DNA were found and the plasmid was highly unstable at 57 degrees C. In strain NUB3621, however, very low amounts of single-stranded plasmid DNA were formed and pTB913-based replicons were only slightly unstable at 57 degrees C. The NUB3621/pTB913 host-vector system seems appropriate for molecular cloning. A RepA-based replicon, also derived from pTB19 but replicating by a theta mechanism, was highly unstable in B. stearothermophilus NUB3621.

DNA Replication↗

PubMedDate not supplied

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development.

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.

Bacteriophages↗