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  • 1
    Electronic Resource
    Electronic Resource
    Cruciform DNA binding protein in HeLa cell extracts (1994)
    s.l. : American Chemical Society
    Biochemistry 33 (1994), S. 14185-14196 
    Details
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1021/bi00251a030
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  • 2
    Electronic Resource
    Electronic Resource
    Chromosomal localization of a sequence with in vivo activity for initiation of DNA replication (1993)
    Springer
    Somatic cell and molecular genetics 19 (1993), S. 103-109 
    Details
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The genomic fragment containing the sequence of human cDNA clone 343, previously characterized as capable of autonomous replication upon transfection into mammalians cells and occupying a genomic region inclusive of an initiation zone for DNA replication, was mapped on human chromosome 6q22-qter by a combination of in situ hybridization and G-banding. Southern blot hybridization with a penl of human-hamster somatic cells confirmed the location of the 343 gene on chromosome 6. Fragile sites have been mapped to the region at 6q21 and 6q26. Several neoplastic disorders, including melanoma, acute nonlymphocytic leukemia, acute lymphocytic leukemia, and malignant lymphoma, have also exhibited translocations and deletions involving the region 6q21–6q27.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01233959
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  • 3
    Electronic Resource
    Electronic Resource
    Defective DNA topoisomerase I activity in a DNAts mutant of Balb/3T3 cells (1985)
    Springer
    Somatic cell and molecular genetics 11 (1985), S. 557-569 
    Details
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Cell and polyomavirus DNA synthesis in ts20, a temperature-sensitive mutant derived from Balb/3T3 cells, is inhibited at an early step in chain elongation in vivo and in vitro. Virus DNA synthesized under restrictive conditions, when analyzed by gel electrophoresis and fluorography, contained a series of equally spaced bands migrating between form I and form II. If restrictive conditions were prolonged, the relative amount of these less-supercoiled topoisomers increased while the overall amount of virus DNA decreased. DNA topoisomerase I activity was lower and more heat-labile when prepared from mutant cells compared to wild-type and revertant cells. An assay in which extracts from wild-type cells corrected defective cell DNA synthesis in lysed mutant cells was applied to purification of the active factor from such extracts. Salt fractionation and three cycles of column chromatography resulted in the isolation of the activity in a fraction containing 10 major polypeptides. The specific activity in the final preparation was increased fivefold and was accompanied by the activity of DNA topoisomerase I. Our results provide evidence that DNA topoisomerase I functions at an early step in chain elongation of cell and polyomavirus DNA synthesis and that the enzyme activity may be decreased as a result of the mutation in ts20.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01534721
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  • 4
    Electronic Resource
    Electronic Resource
    Electron microscopic analysis of in vitro replication products ofors 8, a mammalian origin enriched sequence (1994)
    Springer
    Somatic cell and molecular genetics 20 (1994), S. 147-152 
    Details
    ISSN: 1572-9931
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Electron microscopy was used to map the initiation site of ors 8 DNA replication in vitro in a system that is capable of initiating and supporting one round of semiconservative replication of cloned mammalian DNA origin-enriched sequences (ors). Using unique restriction sites in ors 8 plasmid DNA, we have mapped the replication bubble within the monkey DNA sequence. In addition to site-specific initiation within the ors, the results also indicate bidirectional replication.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02254755
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  • 5
    Electronic Resource
    Electronic Resource
    Conformation of Replicated Segments of Chromosome Fibres in Human S-phase Nucleus (1998)
    Springer
    Chromosome research 6 (1998), S. 595-602 
    Details
    ISSN: 1573-6849
    Keywords: conformation ; replication ; S-phase chromosomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Recent statistical analysis of the folding of G0/G1 chromosomes using fluorescence in situ hybridization (FISH) allowed development of a random walk/giant loop model of chromosome structure. According to this model there are two levels of organization of G0/G1 chromosome fibres. On the first level, the fibres are arranged in giant loops several Mbp in size, and within each loop the fibres are randomly folded. On the second level, the loop attachment sites form a chromosome backbone that also shows random folding. Newly replicated segments of mammalian chromosomes may be directly visualized at high resolution in S-phase nuclei using immunofluorescent methods and appear as worm-like fibres. In our earlier study, we analysed conformation of the fibres in human cells blocked for 16 h at the G1/S boundary with 5- fluorodeoxyuridine (FdU) and then released into S-phase by the addition of a DNA prec ursor. However, long treatment of cells with FdU induces very short replicons and may promote apoptosis. In this study we analysed conformation of the fibres in normally proliferating human cells that had not been blocked with FdU for a long time. It has been found that replicated chromosome fibres visualized just after 2 h of incubation of the cells with a non-radioactively labelled DNA precursor behave as flexible polymer chains without major constraints, and that their local conformation in the range of several microns of their contour length may be considered as random. Confocal analysis of human X chromosomes visualized in HeLa cells using FISH with a specific painting probe shows that in S-phase the chromosomes occupy distinct nuclear territories and their apparent size does not differ from that in non-S-phase cells. This observation indicates that the second level of chromosome organization also exists in S-phase chromosomes. It appears, theref ore, that the random walk/giant loop model developed earlier for G0/G1 chromosomes is also valid for S-phase chromosomes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009293108736
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  • 6
    Electronic Resource
    Electronic Resource
    A reproducible method for identification of human genomic DNA autonomously replicating sequences (1994)
    Springer
    Molecular genetics and genomics 242 (1994), S. 280-288 
    Details
    ISSN: 1617-4623
    Keywords: Origins ; DNA replication ; Human ; In vitro replication assay ; Library screening
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We demonstrate a method for the isolation of autonomously replicating sequences from pools of clones obtained from genomic DNA libraries constructed using affinity purification of cruciform DNA. The selection of autonomously replicating sequences was based on their differential ability to replicate as episomes after transfection of pools of plasmid clones into human HeLa cells. Two separate libraries containing affinity-purified cruciform DNA were used, one prepared from DNA of log phase primary human genital fibroblasts and the other prepared from DNA of log phase SW48 colon adenocarcinoma cells. Representative samples of the entire phage libraries were converted to phagemid clones by filamentous helper phage-mediated mass excision to produce pBluescript libraries in Escherichia coli. Clones were grown up individually and the bacteria pooled into groups of 48 for recovery of plasmid DNA. Plasmid pools of 48 independent clones (120 μg total) were then transfected by calcium phosphate coprecipitation onto log phase HeLa cells, which were allowed to grow for 3 days before recovery of plasmid by Hirt lysis. The recovery of plasmid from each transfection was estimated to range from 10 to 60 ng. DpnI digestion was then used to digest plasmids which had not been replicated and therefore retained a bacterial methylation pattern which was sensitive to digestion. We estimated from agarose electrophesis gels that 40–200 pg of recovered plasmid DNA per transfected pool of DNA was resistant to DpnI and therefore was capable of transforming competent E. coli cells. The DpnI-resistant fraction yielded from one to seven independent clones from each pool, with genomic DNA inserts ranging in size from 0.35 to 3.4 kb. The fidelity of the procedure was demonstrated by performing duplicate transfections from the same pool of plasmid DNA, and identifying bands which were apparently common between duplicate transfections by size and sequence analysis. A second method of mass screening, using an in vitro DNA replication assay instead of transfections, resulted in similar yields and led to the isolation of an overlapping subset of selected clones.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00280417
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  • 7
    Electronic Resource
    Electronic Resource
    Cofractionation of hela cell replication proteins with Ors-binding activity (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 221-236 
    Details
    ISSN: 0730-2312
    Keywords: ors ; replication origin ; replication proteins ; purification ; HeLa cells ; in vitro replication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases α and δ, topoisomerase II, and replication protein A, (RP-A).
    Additional Material: 11 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240580211
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  • 8
    Electronic Resource
    Electronic Resource
    Deletion analysis of minimal sequence requirements for autonomous replication of ors8, a monkey early-replicating DNA sequence (1995)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 57 (1995), S. 280-289 
    Details
    ISSN: 0730-2312
    Keywords: ors ; replication origin ; minimal origin ; deletion analysis ; episomal replication ; in vitro replication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have generated a panel of deletion mutants of ors8 (483 bp), a mammalian autonomously replicating DNA sequence, previously isolated by extrusion of nascent monkey (CV-1) DNA from replication bubbles active at the onset of S phase. The deletion mutants were tested for replication function by the DpnI resistance assay, in vivo, after transfection into HeLa cells, and in vitro. An internal fragment of 186-bp that is required for autonomous replication function of ors8 was identified. This fragment, when subcloned into pBR322 and similarly tested, was capable of autonomous replication in vivo and in vitro. The 186-bp fragment contains several repeated sequence motifs, such as the ATTA and ATTTAT motifs, occurring three and five times, respectively, the sequences TAGG and TAGA, occurring three and seven times, respectively, two 5′-ATT-3′ repeats, a 44-bp imperfect inverted repeat (IR) sequence, and an imperfect consensus binding element for the transcription factor Oct-1. A measurable sequence-directed DNA curvature was also detected, coinciding with the AT-rich regions of the 186-bp fragment.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240570212
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  • 9
    Electronic Resource
    Electronic Resource
    Application of an in vitro system in the study of chemotherapeutic drug effects on DNA replication (1996)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 61 (1996), S. 444-451 
    Details
    ISSN: 0730-2312
    Keywords: in vitro DNA replication ; mammalian ; doxorubicin ; araC ; progesterone ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases α and δ) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication. © 1996 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Deletion analysis of ors12, a centromeric, early activated, mammalian origin of DNA replication (1997)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 66 (1997), S. 87-97 
    Details
    ISSN: 0730-2312
    Keywords: deletion mutants ; ors12 ; replication activity ; mammalian origin ; autonomous replication ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have generated a panel of deletion mutants of ors12 (812-bp), a mammalian origin of DNA replication previously isolated by nascent strand extrusion from early replicating African Green monkey (CV-1) DNA. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the DpnI resistance assay, using extracts from HeLa cells. We identified a 215-bp internal fragment as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and CTTA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats, 22 and 16 bp long, respectively. The overall sequence of the 215-bp fragment is G/C-rich (50.2%), by comparison to the 186-bp (33.5% G/C-rich) minimal sequence required for the autonomous replication activity of ors8, another functional ors that was similarly isolated and characterized. J. Cell. Biochem. 66:87-97, 1997. © 1997 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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