Biomedical subjects

G B Price

Publications and source records attributed to G B Price.

At least 37 records · Page 2Linked to original sources

PubMed1996

Identification of a putative DNA replication origin in the gamma-aminobutyric acid receptor subunit beta3 and alpha5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing.

The region containing the GABAA receptor beta3 and alpha5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone lambda84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage lambda84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13kb of contiguous sequence reveals several A + T-rich regions which display potential DNA unwinding. The plasmid subclones from phage lambda84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABAA receptor subunit gene cluster.

Alleles↗

PubMed1996

Application of an in vitro system in the study of chemotherapeutic drug effects on DNA replication.

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 alpha and delta) 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.

Antibiotics, Antineoplastic↗

PubMed1996

Inverted repeats, stem-loops, and cruciforms: significance for initiation of DNA replication.

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication.

Bacteriophages↗

PubMed1995

Anti-cruciform monoclonal antibody and cruciform DNA interaction.

Cruciform DNA structure, as a structural feature, has been associated with regulation of transcription, recombination and replication. Previously used to successfully modify DNA replication and affinity-purify origins and autonomously replicating sequences. Using enzyme protection assays, their binding activity has been localized to the base (elbow) of the cruciform stem. We report here the hydroxyl radical footprinting of 2D3 (kappa IgG1) anti-cruciform monoclonal antibody on a stable cruciform structure created by heteroduplexing fragments from two plasmids, identical except for two centrally located palindromes of different sequence. The footprinting was performed at near-physiological salt concentrations, conditions favouring the stacked X-structure of the cruciform. Our data show that binding by the antibody occurs at the four-way junction (elbows) of the stable cruciform. The binding of the antibody seems also to cause associated structural distortions in the heteroduplex, which generally result in greater sensitivity to hydroxyl radicals at the tips of the cruciforms. The data are consistent to hydroxyl radicals at the tips of the cruciforms. The data are consistent with the binding of a single antibody to an antigen-combining site. The results of this study compare favourably with the hydroxyl radical footprinting studies reported recently for a human cruciform binding protein (CBP), which binds at the base of the stem-loop structure and causes similar distortions of the stable cruciform structure. These studies indicate that the four-way junction of the cruciform possesses certain unique structural qualities that are antigenic; the association of this structural determinant with DNA replication and the existence of a novel cellular protein, CBP, of similar binding specificity as the antibody specificity support a role for cruciforms as important regulatory recognition signals in replication.

Antibodies, Monoclonal↗

PubMed1995

A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells.

We recently identified and enriched a protein (CBP) from HeLa cells with binding specificity for cruciform-containing DNA. We have now studied the interaction of CBP with stable cruciform DNA molecules containing the 27 bp palindrome of SV40 on one strand and an unrelated 26 bp palindrome on the other strand by hydroxyl radical footprinting. The CBP-DNA interaction is localized to the four-way junction at the base of the cruciforms. CBP appears to interact with the elbows of the junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed in the cruciform stems and in a DNA region adjacent to the junction. These features distinguish CBP from other cruciform binding proteins, which bind symmetrically and display exclusively either contacts with the DNA backbone or structural alterations in the DNA.

Base Sequence↗

PubMed1995

Cofractionation of HeLa cell replication proteins with ors-binding activity.

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 alpha and delta, topoisomerase II, and replication protein A, (RP-A).

Base Sequence↗

PubMed1994

Autonomous replication in vivo and in vitro of clones spanning the region of the DHFR origin of bidirectional replication (ori beta).

Plasmids containing the origin of bidirectional replication (ori beta) of the Chinese hamster dihydrofolate reductase-encoding gene (DHFR) were tested for autonomous replication in vivo and in vitro. The results show that plasmids pX24 and pneoS13, that contain a 4.8- and a 11.5-kb fragment, respectively, spanning the ori beta region, are able to replicate autonomously in human cells and in a cell-free system that uses human cell extracts. Another plasmid, pX14, containing a 4.8-kb fragment that is immediately adjacent to the ori beta region, also replicated in these two assays.

Animals↗

PubMed1994

Cruciform DNA binding protein in HeLa cell extracts.

We have analyzed by band-shift assays HeLa cell protein-DNA interactions on a stable cruciform DNA molecule. The stable cruciform was formed by heteroduplexing the HindIII-SphI fragment of SV40 virus DNA that contains the origin of replication with a derivative mutant containing a heterologous substitution at the central inverted repeat. We have identified a novel binding activity in HeLa cell extracts with specificity for the cruciform-containing DNA and no apparent sequence specificity. The activity is protein-dependent, void of detectable nuclease activity, and distinct from that reported for HMG1. A cruciform binding protein (CBP) with an apparent molecular weight of 66 kDa was enriched from HeLa cell extracts. In addition to the CBP, we have detected sequence-specific binding activities to sites proximal to the cruciform. Binding to one such site is increased in the cruciform-containing heteroduplex DNA by comparison to its linear homoduplex counterpart, suggesting transmission of structural effects by the stem-loops to their local environment.

Base Sequence↗

PubMed1994

Electron microscopic analysis of in vitro replication products of ors 8, a mammalian origin enriched sequence.

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.

Animals↗

PubMed1993

cDNA clones contain autonomous replication activity.

We have undertaken to investigate transcription as a regulatory event in mammalian DNA replication. Subpopulations of transcripts represented in a cDNA library of human embryo lung fibroblasts (IMR90) were examined for their ability to support autonomous replication after transfection into human cells (HeLa). Two of three cDNA clones (343, 363) containing 'O'-family repetitive sequences, after subcloning into pBR322 and transfection into HeLa cells, were capable of autonomous replication. One of these cDNA clones, 343, is enriched by selection for poly(A)+ RNA. In contrast, none of five Alu-containing transcripts was capable of autonomous replication in human cells. However, six out of ten cDNA clones contained neither 'O'-family or Alu homologous sequences and were as efficient as the cDNA clones containing 'O'-family sequences in replicating autonomously in human cells. cDNA clones, from an oligo-d(T)-primed library of human poly(A)+ enriched RNA, contain a significant proportion of independent clones that can also support autonomous replication of bacterial plasmids in human cells. cDNA clone 343 was observed to contain in a 448 bp EcoRI-HincII fragment, yeast ARS consensus, SAR consensus, IRs, bent DNA and a DUE, all sequence and structural characteristics often associated with many prokaryotic, viral and eukaryotic origins. Sequence analysis of seven other cDNA clones (from non-'O'-family, non-Alu homologous sequences, NOA) showed that five contained some of the same consensus sequences. Two NOA clones (NOA4 and -5) did not contain any representations of ARS and SAR consensus sequences, suggesting that these two features may not be essential for autonomous replication activity in mammalian cells.

Animals↗

PubMed1993

In vivo activity for initiation of DNA replication resides in a transcribed region of the human genome.

The potential for autonomous replicating activity has been demonstrated, in the preceding paper, to exist in abundance in those sequences which are transcribed from the genome of human embryonic lung fibroblasts (IMR90). In this paper we demonstrate for one sequence present in an 'O'-family homologous cDNA clone (clone 343), the likelihood of its in vivo activity by analysis of nascent replicated strands. Analysis of seven independent sources of human DNA with three different restriction enzyme digests failed to reveal any significant polymorphisms of the cDNA 343 homologous sequence. Homologous sequences were found in human, cow, mouse and monkey DNA, but not in rat, dog, rabbit, chicken or yeast DNA. An approx. 18 kbp fragment obtained as a clone from a genomic library of human embryonic lung fibroblasts was found to contain the sequence present in cDNA 343 in a 2.2 kbp EcoRI fragment. Sequence analysis of the clone provided three regions suitable for use as oligonucleotide primers in a PCR method of mapping the in vivo site of initiation of DNA replication. The initiation zone and a chromosomal origin for DNA replication are mapped to a region of approximately 1.6 kpb and were inclusive of the sequence detected in cDNA 343.

Base Sequence↗

PubMed1993

Chromosomal localization of a sequence with in vivo activity for initiation of DNA replication.

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 panel 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.

Animals↗

PubMed1993

ors12, a mammalian autonomously replicating DNA sequence, associates with the nuclear matrix in a cell cycle-dependent manner.

Origin enriched sequence ors8 and ors12, have been isolated previously by extrusion of nascent CV-1 cell DNA from replication bubbles at the onset of S-phase. Both have been shown to direct autonomous DNA replication in vivo and in vitro. Here, we have examined the association of genomic ors8 and ors12 with the nuclear matrix in asynchronous and synchronized CV-1 cells. In asynchronously growing cells, ors8 was found to be randomly distributed, while ors12 was found to be enriched on the nuclear matrix. Using an in vitro binding assay, we determined that ors12 contains two attachment sites, each located in AT-rich domains. Surprisingly, in early and mid-S-phase cells, ors12 homologous sequences were recovered mainly from the DNA loops, while in late-S the majority had shifted to positions on the nuclear matrix. In contrast, the distribution of ors8 over the matrix and loop DNA fractions did not change during the cell cycle. By bromodeoxyuridine substitution of replicating DNA, followed by immunoprecipitation with anti-bromodeoxyuridine antibodies and PCR amplification, we demonstrated that ors12 replicates almost exclusively on the matrix in early and mid-S-phase; replicating ors8 was also found to be enriched on the matrix in early S-phase. Chase experiments showed that the ors12 sequences labelled with bromodeoxyuridine in the first 2 hours of S-phase remain attached to the nuclear matrix, resulting in an accumulation of ors12 on the nuclear matrix at the end of the S period.

Animals↗

PubMed1992

Cellular heterogeneity in human transitional cell carcinoma: an analysis of optical properties and lectin binding.

Flow cytometry was used to measure the binding of a panel of ten fluorescein isothiocyanate(FITC)-conjugated lectins to fifteen samples of normal and neoplastic human urothelium. Concurrent measurement of light scattering and fluorescence permitted the quantification of lectin binding to cellular subpopulations defined by their light-scattering properties. In normal urothelium, we previously demonstrated levels of lectin binding to the cellular subpopulations derived from the superficial and intermediate cell layers which were higher than levels which bound to the subpopulation derived from the basal cell layer (Ward et al., 1987). This difference was most marked with Maclura pomifera agglutinin (MPA), Ricinus communis agglutinin (RCA) and Ulex europeus agglutinin (UEA). We now report a similar correlation between the degree of differentiation of a cellular subpopulation and the level of lectin binding in human transitional cell carcinomas (TCCs). Morphological differentiation in human TCCs is accompanied by alterations in cell-surface carbohydrates which are similar to those which accompany cellular differentiation in the corresponding normal tissue. No systematic difference in lectin binding was observed between the corresponding subpopulations of normal and neoplastic urothelial cells.

Carbohydrate Metabolism↗

PubMed1992

ORS12, a mammalian autonomously replicating DNA sequence, is present at the centromere of CV-1 cell chromosomes.

ors12, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. Zannis-Hadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721-727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. Natl. Acad. Sci. USA 84, 6668-6672, 1987) and in a cell-free system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of ors12 (nucleotides 1-168) consists of the highly reiterated alpha-satellite sequence (B. S. Rao et al. Gene 87, 233-242, 1990). We have estimated the copy number of the non-alpha-satellite portion of ors12 in CV-1 cells to be < 9 copies per haploid genome and have used it as a probe to generate a genomic map of ors12 on CV-1 DNA. In situ hybridization of CV-1 metaphase chromosomes, using a biotinylated probe of the entire ors12 sequence, positively identified the centromeres of all chromosomes. However, when the non-alpha-satellite portion of ors12 was used as a probe, it positively identified the centromeric region of only six chromosomes, namely, B4, C11, D14, D24, E25, and E27, as well as that of a marker chromosome. The results suggest that ors12 represents a centromeric putative replication origin that is present on a subset of CV-1 chromosomes and is activated at the onset of S phase.

Animals↗

PubMed1992

Human antiglioma monoclonal antibodies from patients with astrocytic tumors.

The current management of malignant gliomas is unsatisfactory compared to that of other solid tumors; the expected median survival period is less than 1 year with the patient undergoing conventional surgery, radiotherapy, and chemotherapy treatment. Immunological reagents could be a useful adjunct. Human monoclonal antibodies derived from patients with astrocytic tumors might recognize subtle antigenic specificities that would differ from those recognized by xenogeneic (murine) systems. Five hybridomas, designated as BT27/1A2, BT27/2A3, BT32/A6, BT34/A5, and BT54/B8, were produced from the fusion of peripheral blood lymphocytes of four patients with astrocytic tumors to the human myeloma-like cell line TM-H2-SP2. This cell line has a 46, XX karyotype and is negative for hypoxanthine guanine phosphoribosyltransferase. All five human monoclonal antibodies produced 2.4 to 44 micrograms/ml of immunoglobulin M, had a similar but not identical pattern of reactivity against a panel of human tumor cell lines, and failed to react with normal human astrocytes. Labeling of four neuroectodermal tumor explant cultures by BT27/2A3 was demonstrated by flow cytometry. Karyotyping of three of the five hybridomas demonstrated that two were pseudodiploid (2-3n) and one hypodiploid (less than 2n). The monoclonality of the hybridomas was evaluated by Southern blot analysis of JH gene rearrangements, revealing two types of rearrangements for each hybridoma, both consistent with monoclonality. Preliminary antigen characterization indicated that at least four of the five human monoclonal antibodies were directed to cell-surface glycolipids.

Adolescent↗

PubMed1991

DNA cruciforms and the nuclear supporting structure.

Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies with structural specificity for DNA cruciforms have been produced (Frappier et al. J. Mol. Biol. 193, 751, 1987). The effect of these antibodies, when introduced into permeabilized cells, was to increase overall DNA synthesis and relative copy number of genes (Zannis-Hadjopoulos et al. EMBO J. 7, 1837, 1988); this was interpreted to be a consequence of antibody stabilization of the cruciforms located at or near replication origins resulting in multiple initiations of DNA replication at a single site. Fluorescent labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary which then changes with progression through S-phase (Ward et al. Exp. Cell Res. 188, 235, 1990). In order to determine the relationship of cruciform distribution in DNA with the nuclear matrix/chromosomal scaffold, we assessed the susceptibility of DNA containing cruciforms to digestion with DNase I. The majority of the cruciforms detectable at G1/S and throughout the nucleus are readily digested by DNase, suggesting that cruciform structures may not be intimately associated with matrix proteins. The fraction that is resistant to DNase I appears associated with nuclear membrane and the nucleolus. No cruciforms could be detected in metaphase chromosomes; cruciforms either are not present or are inaccessible--buried in the scaffold. The absence of cruciforms from metaphase chromosomes would be consistent with the viewpoint that the cruciform in vivo is a transient structure dependent upon and interacting with proteins essential for replication or transcription.

Animals↗

PubMed1990

Sequence similarities among monkey ori-enriched (ors) fragments.

Nucleotide sequences have been determined for eight ors (ori-enriched sequence) fragments isolated from monkey DNA by a method that was designed to enrich for origins of DNA replication [Kaufmann et al., Mol. Cell. Biol. 5 (1985) 721-727]. Evidence has been presented that some or possibly all of these sequences can serve, albeit inefficiently, as oris in vivo [Frappier and Zannis-Hadjopoulos, Proc. Natl. Acad. Sci. USA 84 (1987) 6668-6672]. Two of the fragments were found to contain the long terminal repeat-like elements of the 'O-family' of moderately repetitive sequences that are present in human DNA as a transposon-like element [Paulson et al., Nature 315 (1985) 359-361]. Extensive pair-wise comparisons of the sequences failed to detect any statistically significant common sequences, except for long asymmetrically distributed A + T-rich stretches. Nonetheless, when the ors fragments were examined for the presence of published consensus sequences, seven of eight were found to contain the control sequence described by Dierks et al. [Cell 32 (1983) 695-706], and the same seven of eight were found to contain both the scaffold attachment region T consensus [Gasser and Laemmli, Cell 46 (1986) 521-530] and the minimal Saccharomyces cerevisiae autonomously replicating sequence consensus [e.g., Palzkill and Newlon, Cell 53 (1988) 441-450].

Amino Acid Sequence↗