Biomedical subjects

S Scherneck

Publications and source records attributed to S Scherneck.

At least 73 records · Page 4Linked to original sources

PubMed1985

Characterization of the DNA of the hamster papovavirus: II. A comparison of binding sites for Escherichia coli- and calf thymus RNA polymerase II on the hamster papovavirus genome.

Calf thymus (CT) RNA polymerase II bound to hamster papovavirus (HaPV) DNA was visualized by electron microscopy and compared to binding sites obtained after binding of Escherichia coli RNA polymerase to the HaPV genome. Thirteen binding sites were observed with CT polymerase II at map positions 0.04-0.07; 0.11; 0.18; 0.30; 0.37; 0.47; 0.57; 0.65; 0.78; 0.83; 0.90 and 0.94 using the unique BamHI cleavage site as zero point on the HaPV physical map. These binding sites correlate well with A + T rich sequences within the HaPV DNA as revealed by experiments using protein 32 coded for by phage T4. A comparison of binding of prokaryotic and eukaryotic RNA polymerase demonstrates a high degree of correspondence for most of the binding sites on the HaPV genome.

Animals↗

PubMed1985

Heterogeneity, molecular weight and stability of an oncogenic papovavirus of the Syrian hamster.

Sedimentation, isopycnic density gradient and molecular weight studies of an oncogenic papovavirus of the Syrian hamster have been performed. The sedimentation coefficient of 223 S, the buoyant density of 1.340 g/ml, and the molecular weight of 27.5 X 10(6) are in close correspondence with those of SV 40 (simian virus 40) and polyoma viruses. Deviating from these members of the papovavirus group, a sedimentation heterogeneity resulting in the presence of dimers and higher virion oligomers up to pentamers besides heterogeneous material of viral origin was demonstrated by sedimentation velocity. Also, a microheterogeneity of the buoyant density of the virus was demonstrable which is discussed to reflect this virion association. The stability of hamster papovavirus was examined after exposure to alkaline pH and the virus destabilizing agents dithiothreitol and ethyleneglycol-bis-N,N'-tetraacetic acid. In neutral conditions a nucleoprotein complex of 170 S was demonstrated. Further disintegration of this complex with increasing pH resulted in its conversion to nucleoprotein complexes of 125 S and 90 S in analogy to SV 40 and polyoma viruses.

Animals↗

PubMed1985

Restriction endonuclease cleavage map of the DNA of hamster papovavirus.

A detailed restriction map of hamster papovavirus (HaPV)-DNA has been constructed on the basis of the complete nucleotide sequence of the viral genome. The HaPV DNA restriction map is totally divergent from those of all other recently known papovaviruses. This map will be useful for future functional studies of the HaPV genome.

Animals↗

PubMed1984

Interaction of histone H1 with superhelical DNA. Conformational studies and influence of ionic strength.

The interaction of histone H1 with superhelical SV40 DNA at low ionic strength (approximately 0.02 M NaCl) results in the formation of DNP double-fibers and bundle- and cablelike twisted side-by-side associates of several of these double-fibers. On the basis of simple cylindrical or ellipsoidal models the sedimentation properties of these structures can be calculated in accordance with the experiment allowing a direct assignment of electron microscopical and hydrodynamic results. Sedimentation measurements in dependence on the ionic strength indicate a redistribution of H1 resulting in the formation of associates at 0.04 M NaCl and of aggregates at higher salt concentration. Double-fibers are present up to physiological salt concentrations.

DNA, Superhelical↗

PubMed1984

Sequence homology between polyoma virus, simian virus 40, and a papilloma-producing virus from a Syrian hamster: evidences for highly conserved sequences.

Sequence homology between the genomes of a hamster papovavirus (HaPV), polyoma virus (Py), and Simian virus 40 (SV40) has been studied by filter hybridization and electron microscopy under conditions of varying stringency. Hybrids between the HaPV and SV40 DNAs could be demonstrated only under nonstringent conditions. The region of highest homology was mapped in the early region of the SV40 genome. Extensive homology was detected between the genomes of HaPV and Py under stringent hybridization conditions, indicating at least 80% base matching in the regions of strongest sequence homology. These sequences were localized within both the early region and the late region of the Py genome. The homologous DNA segments mapped in the Py and the SV40 genomes are among the most strongly conserved regions in the polyoma (miopapova)-virus group.

Animals↗

PubMed1984

Characterization of the DNA of the hamster papovavirus: I. Genom length and molecular cloning.

The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of the Syrian hamsters was measured by electron microscopy and cloned in Escherichia coli using the certified plasmid vector pBR322. The cloned viral DNA were characterized by digestion of the recombinant DNA with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNA and by electron microscopy to determine the genome size of cloned HaPV DNA. The restriction enzyme analysis of the cloned HaPV DNA showed the same cleavage pattern as the corresponding fragments from the uncloned DNA. No major insertions or deletions could be detected by heteroduplex analysis between cloned HaPV DNA and the starting material. The estimated genome size of 5.52 kb for HaPV DNA is approx. 300 bases larger than those determined for other known papovaviruses as SV40 or polyoma.

Animals↗

PubMed1983

Studies on the SV40-like papovavirus SV40-GBM. II. Molecular cloning in Escherichia coli of variant DNA molecules from glioblastoma-derived virus.

The complete DNA genomes of the SV40-like GBM virus (GBM1), isolated from a human glioblastoma multiforme, and of two discrete classes of GBM DNA molecules that appear following three passages in CV-1 monkey cells at low multiplicities (GBM3-H and GBM3-L), were cloned in Escherichia coli using plasmid vector pBR322. The cloned viral DNAs were characterized (i) by digestion of the chimeric plasmid DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose with that of similarly digested uncloned DNA, (ii) by hybridization of digested chimeric plasmid DNAs to 32P-labeled uncloned GBM DNA, and (iii) by electron microscopy. In restriction enzyme analysis the cloned GBM DNAs showed the same cleavage pattern as the uncloned DNAs, indicating that no major insertions or deletions were present. The electrophoretic data were confirmed by electron microscopic heteroduplex analysis.

Cloning, Molecular↗

PubMed1983

Studies on the SV40-like papovavirus SV40-GBM. I. Genomic analysis by restriction endonucleases and electron microscopy after propagation in CV-1 monkey cells.

Infection of CV-1 monkey cells with SV40-GBM, a papovavirus isolated from a human glioblastoma multiforme, resulted in the appearance of defective viral DNA molecules. In contrast to SV40 wild-type, two main types of variant DNA molecules could be found after three viral passages at multiplicities of infection of about 10. The molecules of one variant DNA (GBM3-L) were about 19% shorter than the GBM3-H DNA molecules and the DNA of the original GBM isolate, as demonstrated by electron microscopy. Restriction enzyme analysis revealed that GBM3-L DNA had lost both the EcoRI and the HpaII cleavage sites which are located in the late viral genome region. Furthermore, SV40 GBM3-L did not possess the two PvuII sites which are located in the late genome region, and a portion of the GBM3-H and GBM3-L DNA molecules had lost the unique KpnI site. Heteroduplex analysis verified that the rearrangements in the GBM3-L DNA are located only in the late region of this DNA. The possible differences between SV40 wild-type and SV40-GBM are discussed on the basis of these results.

Animals↗

PubMed1983

In vitro transformation and mutation of Chinese hamster cells by different SV40 nucleoprotein complexes.

SV40 minichromosomes (MCH) either isolated from SV40 infected CV-I monkey cells (native MCH) or reconstituted in vitro from viral DNA and the H1 depleted calf thymus histone fraction could transform and mutate Chinese hamster (CH) cells in vitro. Whereas reconstituted MCH transformed and mutated CH cells with about the same efficiency as purified SV40 DNA, approximately 10-200-fold increase in the transforming activity had been demonstrated for native MCH. All transformed cell colonies and a major part of the isolated mutant cell clones recovered after inoculation of CH cells with SV40 MHC expressed the SV40 T antigen. Addition of H1 to both purified SV40 DNA and reconstituted MHC drastically diminished the transforming capacities of both agents. Possible reason(s) for the inhibition effect of H1 histone is discussed.

Animals↗

PubMed1983

Immortalization of human Lesch-Nyhan-fibroblasts following infection with Simian virus 40.

Skin fibroblasts of three Lesch-Nyhan patients were successfully transformed to unrestricted growth by Simian virus 40. The transformed character of isolated clones was confirmed by their epitheloid growth in medium containing low concentration of serum, by hyperdiploid karyotype patterns, by detection of SV40 T-antigen, by their growth in soft agar and by the proliferation of cells in vitro up to 200 passages hitherto.

Animals↗

PubMed1983

Evolutionary relationships between papovaviruses and their hosts.

The papovaviridae family consists of two genera, the papillomaviruses (PV) and the polyomaviruses (Py-V). Both genera are distinguished by morphological (larger sizes of the PV) and several biological characteristics. The genomes of either of the two genera share highly conserved DNA regions and a common antigenic determinant, located in their major capsid polypeptides. On the basis of these data an evolutionary relationship among the members of PV and Py-V, respectively, has been suggested. No homology has been found for either DNA- or protein sequences between PV and Py-V and the question of a common ancestor for both viral genera remains open. We have started to characterize the genome of a papilloma producing papovavirus of the Syrian hamster (HaPV). Most of the known biological characteristics of the HaPV suggest it should be classified as a papilloma-like virus. However, the molecular weight of about 3.5 X 10(6) daltons found for the circular duplex DNA lies within the range given for SV 40 and polyoma virus (Py). Analysis of the HaPV genome by cleavage with 21 different restriction endonucleases, location of specific binding sites of phage T 4 gene 32 protein and E. coli RNA polymerase on the viral DNA demonstrated that the HaPV differed distinctly from all other currently known papovaviruses. The HaPV genome was also analyzed by filter hybridization and electron microscopy under conditions of varied stringency for nucleotide sequence homology with the genomes of different papovaviruses of both genera. Whereas no homologous DNA regions could be found between the genomes of HaPV and the human PV types 1 and 4, only under nonstringent conditions (Tm-43 degrees C) stable hybrids were formed between HaPV-, SV 40- and the DNA of a PV isolated from Mastomys natalensis (MnPV). On the other hand extensive homology was detected between the genomes of HaPV and Py even under stringent hybridization conditions (Tm-28 degrees C). The homologous DNA segments mapped on the Py and partially on the SV 40 genome were found to be the most strongly conserved DNA regions among the Py-V genus. These results are discussed with respect to a classification of the HaPV within the papovaviridae family.

Animals↗

PubMed1983

Are transforming and mutagenic activities of oncogenic papovaviruses correlated?

A number of results obtained recently have shown that SV40-induced mutation and transformation of mammalian cells cultivated in vitro are related in some respect. Experiments were undertaken in order to get further information on the mode of mutagenic action of papovaviruses and further evidence of correlations existing between the mutagenic and transforming viral activities. DNA from the oncogenic hamster papovavirus HaPV was found to be mutagenic for at least three different resistance markers. In the hamster cell lines which were used HaPV DNA produced a higher yield of mutants than SV40 DNA for both the azaguanine and the aminopterin resistance marker. Cellular clones carrying a SV40-induced mutation in a specific locus were found to harbour viral genetic material and to exhibit a genetic instability of other loci. Also, cell lines characterized by their SV40-transformed state did exhibit such genetic instability. These results and results of other authors are discussed with respect to the correlations between oncogenic virus-induced mutation and transformation.

Animals↗

PubMed1983

Quantitative determination of papovavirus IgG antibodies in sera from cancer patients, labworkers and several groups of control persons by enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) for detection of specific IgG antibodies against the capsid antigen of SV40 and SV40-GBM is described. We have determined the presence of SV40 and SV40-GBM IgG antibodies, respectively, in sera of labworkers who are in intensive or rare contact to SV40, in sera of tumor patients as well as in sera of different control persons. In 13% of the sera of the control group antibodies against SV40 were found, when the persons were born 1962 or later. They received SV40-free poliomyelitis vaccine. On the other hand 24% of the sera were positive when the people were born between 1959-61 and probably received SV40-contaminated poliomyelitis vaccine. A similar percentage of positive sera (21-32%) were found in cancer patients suffering on different tumors. Also 29% of the sera of labworkers, born before 1955 and with rare contact to SV40 reacted positive. However, 55% of the sera of labworkers had positive sera when they were in intensive contact to SV40. The results presented and discussed in the light of SV40 as a possible risk factor for the human population.

Adult↗

PubMed1982

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes.

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native 'minichromosomes'. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.

Animals↗

PubMed1982

A comparison of the phage T4 gene 32 protein and Escherichia coli RNA polymerase binding sites on hamster papovavirus DNA.

Phage T4 gene 32 protein and Escherichia coli RNA polymerase were bound to hamster papovavirus DNA. The binding regions were identified by electron microscopy employing a protein-free spreading technique. After gene 32 protein treatment four denaturation regions could be mapped, at 0.04-0.12, 0.30-0.36, 0.50-0.60 and 0.75-0.90 DNA map units, respectively, using the unique BamHI cleavage site as zero point. Eight RNA polymerase binding sites can be found which are localized at positions 0.05; 0.11; 0.18; 0.31; 0.57; 0.66; 0.76 and 0.82. A comparison of the RNA polymerase binding sites with the gene 32 protein denaturation pattern reveals a correspondence of six of eight polymerase binding sites with (A+T)-rich regions within the hamster papovavirus genome.

Binding Sites↗

PubMed1982

The DNA tumor virus SV 40 induces gene mutations in human cells. Reversion of HPRT deficiency.

The mutagenic effect of papovavirus SV40 on human cells could be demonstrated by reversion of HPRT deficiency in Lesch-Nyhan fibroblasts transformed by the virus. SV40 seems to induce different gene mutations in individually selected cell clones, as was clearly shown by the respective HPRT enzyme properties. The consequences of our results for use of SV40-derived vectors in gene substitution experiments are discussed.

Cell Transformation, Viral↗

PubMed1981

Interaction of histone H1 with superhelical DNA. Sedimentation and electron microscopical studies at low salt concentration.

Complexes of histones H1 with superhelical SV40 DNA obtained by direct mixing were studied in 0.1 SSC buffer corresponding to 0.02 M Na+. Depending on the molar input ratio H1/DNA three classes of sedimenting species were observed: (1) a component sedimenting similar to superhelical DNA with a sedimentation coefficient s2o,w of 25 S observable up to 335 Mol H1/Mol DNA (w/w = 2); (2) a component with s2o,w = 120 S appearing at 135 Mol H1/Mol DNA and (3) growing amounts of heterogeneous aggregates greater than 1000 S. Electron micrographs revealed the 25 S component to consist of double-fibers formed from one DNA molecule and the 120 S component to consist of bundles of several such double-fibers. The aggregates represent cable-like structures. The addition of ethidium bromide to 25 S complexes induces the formation of bundles, if H1 is present in a quantity which alone is not sufficient to bring about this effect. This result indicates that ethidium bromide effects a redistribution of H1 molecules and that H1 is responsible for the bundle formation.

DNA, Superhelical↗

PubMed1981

Influence of superinfection on the photoreversible phase of UV induced lysogenic bacteria.

1. Lysogenic induction by UV light can be reversed by photoreactivation. UV-treated E. coli K12 (lambda)+ uvr+ and uvr cells are sensitive to photoreactivation for a given time after irradiation. This sensitivity suddenly disappears at the end of this time. 2. The photoreversible period of UV induction is more than twice as long in uvr cells as it is in uvr+ cells. 3. The photoreversible period can be reduced by superinfection with lambda c mutants after irradiation. This effect is positively correlated with the multiplicity of superinfection. Such a reduction does not occur when superinfection is carried out with wild-type phages or with heteroimmune derivatives. 4. We concluded that during the photoreversible period of UV induction oligonucleotides are excised or synthesized and gaps are formed during excision repair and post replication repair of UV damage; these might react with E. coli recA protein thereby activating it to induce its own synthesis, to cleave phage repressors and to exert its other SOS functions.

Bacteriophage lambda↗