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PubMed1984

Isolation of short interspersed repetitive DNA sequences present in the regions of initiation of mammalian DNA replication.

Nascent DNA chains containing the putative replication origins were isolated from cells of human embryonic lung fibroblasts, Hela, Ehrlich ascites tumour and Guerin ascites tumour as described earlier [ Russev , G., and Vassilev , L. (1982) J. Mol. Biol. 161, 77-87]. It was demonstrated that the synthesis of these nascent chains correlated with the ability of cells to initiate semiconservative DNA replication. Reassociation and electrophoretic analysis showed that the nascent chains from all four cell lines contained middle repetitive DNA in the form of short interspersed sequences. Mouse repetitive sequences were isolated and hybridized to Escherichia coli, chicken, calf and rat DNA and to homologous hnRNA. The kinetics of hybridization indicated that the repetitive sequences found in the vicinity of the replication origins were order-specific and were not heavily transcribed. Reassociation experiments, in which homologous DNA isolated from nuclei digested with micrococcal nuclease to different extents was used as a driver, showed that these repetitive sequences were organized into nucleosomes like the bulk of the chromatin.

Animals↗
PubMed1993

Mitochondrial RNA abundance in differentiating human colonic epithelial tumor cells estimated through use of a mitochondrial genome map.

We describe a procedure for quantification of mitochondrial (mt) RNA present in total RNA extracts of HT-29 human colonic adenocarcinoma cells grown under conditions for rapid growth (25 mM glucose) or differentiation (25 mM trehalose or 5 mM butyrate). Purified mt DNA was fragmented into specific coding regions using restriction endonuclease sites predicted from HeLa cell mt DNA sequence and probed with either 32P-labelled mt DNA or cDNA made from total RNA of HT-29 or HeLa cells. The amounts of probe that hybridized to various gene-encoded mt DNA fragments or RNA were quantified by laser densitometry. Use of 13 restriction endonucleases revealed that most if not all the mt DNA of HT-29 and K562 leukemic cells was comparable in size to that of HeLa cells. Relative levels of mt RNA from rapidly growing HT-29 and HeLa cells were lower than those measured for differentiated HT-29 cells induced by either trehalose or butyrate. In rapidly growing HT-29 cells and HeLa cells, the highest levels of specific mt RNAs were those encoded by mt DNA sequences immediately flanking the nested promoters and the heavy-strand replication origin (OH). Expression patterns of specific mt RNAs from HT-29 cells treated with butyrate and with trehalose were similar, but not identical. In either case, the mt RNAs that increased the most were those coded by mt DNA sequences located downstream from the light-strand replication origin (OL), suggesting a novel pattern of expression not seen before.

Butyrates↗
PubMed1990

Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.

Animals↗
PubMed1993

Characterization of human papillomavirus type 11 E1 and E2 proteins expressed in insect cells.

The study of human papillomavirus replication has been hampered by the lack of an in vitro system which reliably supports virus replication. Recent results from the bovine papillomavirus (BPV) system indicate that the E1 and E2 proteins are the only viral gene products required for replication. By analogy with simian virus 40 large T antigen, E1 is thought to possess ATPase and helicase activity, which may play a direct role in viral DNA replication. The precise role of E2 is unclear, but it may function in part to help localize E1 to the replication origin. We have initiated a study of replication in the human papillomavirus type 11 system which, by analogy to BPV, has focused on the E1 and E2 proteins of this virus. We have expressed the full-length E1 and E2 proteins in Sf9 insect cells by using a baculovirus expression vector. Both the 80-kDa E1 protein and the 42.5-kDa E2 protein are nuclear phosphoproteins. The E1 and E2 proteins form a heteromeric complex within the insect cells, and both proteins localize to a DNA fragment which contains the viral origin of replication. In addition, we have detected an E1-associated ATPase and GTPase activity, which is likely part of an energy-generating system for the helicase activity which is predicted for this protein. The human papillomavirus type 11 E1 and E2 proteins possess the same replication-associated activities exhibited by the corresponding BPV proteins, suggesting that the replication activities of these viruses are tightly conserved.

Adenosine Triphosphatases↗
PubMed1998

A rolling circle replication initiator protein with a nucleotidyl-transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi.

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking-closing activity in vitro on single-stranded oligonucleotides containing the putative double-stranded replication origin sequence of pGT5. Gel shift analyses on single-stranded oligonucleotides indicate that Rep75 recognizes the single-stranded DNA region upstream of the nicking site via non-covalent interaction and remains covalently linked to the 5'-phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3'-OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.

Bacterial Proteins↗
PubMed1984

Distribution of "intrastrand annealing" sequences in lambda bacteriophage genome.

Our computer programme which is originally developed for calculating distribution of folding structures in single stranded nucleic acids was applied for analysis of lambda phage DNA. We found that we have to choose an optimum condition of calculation depending upon what kind of structures we are looking for, for example secondary structure of transcripts or clover-leaf structures in the double stranded DNA molecules. In one calculation condition, the lambda genome was divided into two distinct regions, i.e., capsid genes with high folding probabilities. In this condition, the clover-leaf structure previously indicated in the replication origin was not detected. Calculation in the condition which included annealings with higher free energies (lower stabilities) revealed the presence of the clover-leaf structure in the DNA replication origin in the middle of the gene O.

Bacteriophage lambda↗
PubMed2000

Start sites of bidirectional DNA synthesis at the human lamin B2 origin.

The initiation sites of bidirectional synthesis at the DNA replication origin located at the 3' end of the human lamin B2 gene were investigated. RNA-primed nascent DNA molecules were subjected to second-strand synthesis with appropriate primers, amplified by ligation-mediated polymerase chain reaction, and size fractionated. Evidence for precise start sites was obtained. Exploration of close to 1 kilobase, coupled to inhibition of Okazaki fragment synthesis, demonstrates that the leading strands initiate at precise nucleotides on either helix, overlapping by three base pairs, within the area bound to a protein complex possibly analogous to the prereplicative complex of yeast.

AT Rich Sequence↗
PubMed1997

Stringent structural and sequence requirements of the human herpesvirus 6B lytic-phase origin of DNA replication.

The lytic-phase origin of DNA replication from human herpesvirus 6B (HHV-6B oriLyt) contains two binding sites for the origin-binding protein (OBPH6B), both of which are required for DNA replication and which are separated by an AT-rich spacer. We have tested the functional significance of the structural, spatial and sequence characteristics of this spacer element by constructing a series of mutated origin sequences and analysing their replication efficiency. Changes in the sequence composition of length of the spacer resulted in dramatic decreases in replication efficiency. Furthermore, in contrast to what has been observed for herpes simplex virus type 1 (HSV-1) oriS, insertion of a complete helical turn of DNA into the spacer also resulted in abrogation of origin function. These data suggest that the arrangement of OBP sites in HHV-6B oriLyt is stringently constrained in terms of spacing and intervening sequence.

Base Sequence↗
PubMedDate not supplied

Construction of a plasmid vector for the regulatable high level expression of eukaryotic genes in Escherichia coli: an application to overproduction of chicken lysozyme.

A novel expression vector pKP1500 for synthesizing unfused protein in Escherichia coli was constructed. pKP1500 perserves the tac promoter, the lacZ SD sequence, unique restriction sites (EcoRI, SmaI, BamHI, SalI, PstI and HindIII) and the rrnB terminators of pKK223-3, but the replication origin is replaced with that of pUC9. Construction of this plasmid is based upon the observation that the copy number control of pUC9 is temperature dependent. At 28 degrees C, the copy number of pKP1500 is less than 25 per chromosome, approximately the same copy number as that of pKK223-3, which contains the replication origin of pBR322, whereas at 42 degrees C, the copy number increases about 10 times and reaches up to 230 copies per chromosome. The main advantage of this system is that the temperature-dependent copy control and regulatable expression of the tac promoter make cells carrying pKP1500 derivatives stable against selective pressure by detrimental overproduction of foreign proteins at a low temperature and permits high expression of cloned DNAs at a high temperature. When chicken lysozyme cDNA carrying the initiation codon (ATG) immediately upstream from the Lys1 codon was inserted downstream from the tac promoter and the SD sequence, the pKP1500 derivative produced lysozyme at about 25% of the total cellular proteins. This value is more than 10 times higher than that obtained with the pKK223-3 derivative carrying the same lysozyme cDNA. By comparison, the expression of eukaryotic genes from the tac promoter reported by others has usually been less than a few % of the total cellular protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

The Cdc7 protein kinase is required for origin firing during S phase.

The Cdc7p protein kinase plays an essential, but undefined, role promoting S phase in the budding yeast, Saccharomyces cerevisiae. Previous experiments have shown that the essential function of Cdc7 is executed near the G1-S boundary; after Start but before the elongation phase of DNA replication. Origins of DNA replication fire throughout S phase in budding yeast. Therefore, the G1-S transition is a cell-cycle event that precedes, and is distinct from, the activation of individual origins. Consequently, we have asked whether Cdc7 is only required for S-phase entry or if it plays a role during S phase in origin firing. In this article, we show that partial loss of Cdc7 function results in slow progression through S phase rather than slow entry into S phase and that Cdc7 is still required for the timely completion of S phase after a block to elongation with hydroxyurea. This is because Cdc7 is still required for the activation of late-firing origins after the hydroxyurea block. These experiments show that, rather than acting as a global regulator of the G1-S transition, Cdc7 appears to play a more direct role in the firing of replication origins during S phase.

Cell Cycle Proteins↗
PubMed1999

Construction of Epstein-Barr virus-based expression vector containing mini-oriP.

Epstein-Barr virus (EBV)-based vectors are extrachromosomal vectors carrying a replicational origin, oriP (about 2200 bp) and a replication initiation factor (EBNA-1) which are sufficient for autonomous replication. Because one disadvantage of these vectors is their large sizes, we examined the effect of partial deletion of oriP on the effectiveness of the EBV-based vectors, using an enhanced green fluorescent protein (EGFP) as a reporter to monitor gene expression. Results indicated that 954 bp-deleted mini-oriP is useful in primate cells since the vector showed high efficiency of stable transfection, a high ratio of EGFP-positive cells, and high recovery of intact plasmid DNA from transfected cells.

Animals↗
PubMed2001

DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum.

Two-dimensional neutral/neutral agarose gel electrophoresis is used extensively to localize replication origins. This method resolves DNA structures containing replication forks. It also detects X-shaped recombination intermediates in meiotic cells, in the form of a typical vertical spike. Intriguingly, such a spike of joint DNA molecules is often detectable in replicating DNA from mitotic cells. Here, we used naturally synchronous DNA samples from Physarum polycephalum to demonstrate that postreplicative, DNA replication-dependent X-shaped DNA molecules are formed between sister chromatids. These molecules have physical properties reminiscent of Holliday junctions. Our results demonstrate frequent interactions between sister chromatids during a normal cell cycle and suggest a novel phase during DNA replication consisting of transient, joint DNA molecules formed on newly replicated DNA.

Animals↗
PubMed1997

Cdc6p establishes and maintains a state of replication competence during G1 phase.

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

Cell Cycle Proteins↗
PubMed1988

Chloroplast DNA replication in vitro: site-specific initiation from preferred templates.

An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17' (2470 bp) and Eco RI x (1368 bp) have been shown to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194-198]. Analysis of replicative intermediates indicates that although the template activity of the recombinant plasmid pZmcBam 17' is substantially greater than that of the pZmcEco x, replication in both cases originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion of maize chloroplast BamHI fragment 17' is not more active because it contains additional origins for replication. The overlapping region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455 bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA insert, a region that includes the 3' terminal part of the psbA gene.

Chloroplasts↗
PubMed2001

Sna41goa1, a novel mutation causing G1/S arrest in fission yeast, is defective in a CDC45 homolog and interacts genetically with polalpha.

Proteins involved in the initiation of DNA replication play critical roles in the assembly and loading of replication complexes at replication origins. To gain further insight into the regulation of initiation, we screened in fission yeast for temperature-sensitive mutants which arrested at the G1/S boundary, and isolated nine mutants which arrested with a 1C DNA content at 36 degrees C. By linkage analysis, two complementation groups were identified which were not allelic to known G1 arrest mutations. One of the mutants isolated, sna41goul, arrested with a G1 DNA content and expressed a pleiomorphic phenotype, i.e., a mixture of cut and cdc phenotypes, at 36 degrees C. The point of arrest was identified as after START but before the hydroxyurea-induced block, by taking advantage of the mutant rad26.a14, which has a defect in an early S phase-specific checkpoint, and by performing reciprocal shift experiments. sna41 goal is allelic to sna41+, which is homologous to the CDC45 gene of budding yeast, and the mutation lies in a motif that is highly conserved in Cdc45-related proteins. The temperature sensitivity of the sna41goal mutant can be suppressed to some extent by ts mutations in polalpha. Our genetic results are consistent with a model in which Cdc45 plays crucial roles in the assembly of the replication apparatus at replication origins.

Carrier Proteins↗
PubMed1998

Improvement of retroviral packaging cell lines by introducing the polyomavirus early region.

To obtain high-titer recombinant retroviruses, we constructed plasmid pDL+, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL+ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, psi2 and PA317, by stably introducing the polyomavirus early gene, and established psiMP34 and psiMP37 from psi2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10-100 times higher than produced by the parent cell line and reached levels of 0.5-1.5 x 106 cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 x 10(7) cfu/ml from psiMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome.

3T3 Cells↗
PubMed1989

Breakage--reunion and copy choice mechanisms of recombination between short homologous sequences.

To study recombination between short homologous sequences in Escherichia coli we constructed plasmids composed of the pBR322 replicon, M13 replication origin and a recombination unit inserted within and inactivating a gene encoding chloramphenicol resistance. The unit was composed of short direct repeats (9, 18 or 27 bp) which flanked inverted repeats (0, 8 or 308 bp) and a gene encoding kanamycin resistance. Recombination between direct repeats restored a functional chloramphenicol resistance gene, and could be detected by a simple phenotype test. The plasmids replicated in a double-stranded form, using the pBR322 replicon, and generated single-stranded DNA when the M13 replication origin was activated. The frequency of chloramphenicol-resistant cells was low (10(-8)-10(-4] when no single-stranded DNA was synthesized but increased greatly (to 100%) after induction of single-stranded DNA synthesis. Recombination between 9 bp direct repeats entailed no transfer of DNA from parental to recombinant plasmids, whereas recombination between 18 or 27 bp repeats entailed massive transfer. The presence or length of inverted repeats did not alter the pattern of DNA transfer. From these results we propose that direct repeats of 9 bp recombine by a copy choice process, while those greater than or equal to 18 bp can recombine by a breakage-reunion process. Genome rearrangements detected in many organisms often occur by recombination between sequences less than 18 bp, which suggests that they may result from copy choice recombination.

Base Sequence↗
PubMed1998

Chromosome arrangement within a bacterium.

BACKGROUND: The contour length of the circular chromosome of bacteria is greater than a millimeter but must be accommodated within a cell that is only a few micrometers in length. Bacteria do not have nucleosomes and little is known about the arrangement of the chromosome inside a prokaryotic cell. RESULTS: We have investigated the arrangement of chromosomal DNA within the bacterium Bacillus subtilis by using fluorescence microscopy to visualize two sites on the chromosome simultaneously in the same cell. Indirect immunofluorescence with antibodies against the chromosome partition protein Spo0J were used to visualize the replication origin region of the chromosome. Green fluorescent protein fused to the lactose operon repressor Lacl was used to decorate tandem copies of the lactose operon operator lacO. A cassette of tandem operators was separately inserted into the chromosome near the origin (359 degrees), near the replication terminus (181 degrees), or at two points in between (90 degrees and 270 degrees). The results show that the layout of the chromosome is dynamic but is principally arranged with the origin and terminus maximally apart and the quarter points of the chromosome in between. CONCLUSIONS: The use of cytological methods to visualize two chromosomal sites in the same cell has provided a glimpse of the arrangement of a bacterial chromosome. We conclude that, to a first approximation, the folding of the bacterial chromosome is consistent with, and may preserve, the linear order of genes on the DNA.

Bacillus subtilis↗