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H Probst

Publications and source records attributed to H Probst.

13 recordsLinked to original sources
PubMed1992

Reversible shutdown of replicon initiation by transient hypoxia in Ehrlich ascites cells. Dependence of initiation on short-lived protein.

The O2-dependent regulation of replication in Ehrlich ascites cells, characterized by a reversible shutdown of replicon initiation during hypoxia, was scrutinized with respect to the involvement of gene expression. Synchronous and asynchronous cells were subjected to transient hypoxia and examined for expression of selected 'late' growth-regulated mRNA and for the influence of inhibitors of transcription and translation on DNA replication. Irrespective of whether replicon initiation was suppressed by hypoxia or retriggered by reoxygenation, the levels of thymidine kinase mRNA and of proliferating cell-nuclear antigen/cyclin mRNA were as high as in untreated replicating cells. The level of histone H3.1 mRNA followed, with a distinct delay, the replicative activity of the cells governed by the imposed changes of pO2. The response of replication to inhibition of transcription and translation was virtually the same as to hypoxia, i.e. a selective suppression of replicon initiation. It was demonstrated that replicon initiation depends on one or several short-lived protein(s) (lifetime about 5 min) which is (are) formed under hypoxic conditions as well. The lifetime of the corresponding RNA message(s) is in the range of several hours. It is suggested that the expression of genes conditioning resting cells for DNA replication remains unaffected by hypoxia or by restoring the normal pO2. Hypoxic cell appear to rest in a state fully prepared for entering DNA replication, but a yet unknown event essential for replicon initiation is blocked. This event depends on a critical oxygen tension as well as on short-lived protein(s).

Animals↗
PubMed1992

Cycloheximide inhibits cellular, but not SV40, DNA replication.

We have prepared extracts from cycloheximide-treated cells for the study of simian-virus-40 (SV40)-DNA replication in vitro. When supplemented with the viral initiator protein (large T antigen), these extracts fully supported SV40-DNA replication. We also determined that SV40-DNA replication in vivo is much more resistant to cycloheximide than cellular DNA replication. SV40 encodes its own initiator protein, T antigen, which also functions as a DNA helicase, but depends on cellular functions for all additional replication reactions. Therefore, it appears to be quite likely that cycloheximide affects cellular DNA replication by blocking the synthesis of (a) cellular function(s) that is(are) performed by T antigen in SV40-DNA replication. Indeed, DNA fiber autoradiography and alkaline sucrose gradient centrifugation of pulse-labeled cellular DNA showed that cycloheximide treatment almost completely suppressed replicon initiation and reduced the rate of replication fork movement to about one third of the control.

Antigens, Polyomavirus Transforming↗
PubMed1990

mdr1/P-glycoprotein gene segments analyzed from various human leukemic cell lines exhibiting different multidrug resistance profiles.

Three high-level multidrug-resistant sublines of the human T-lymphoblastoid cell line CCRF-CEM were selected independently with either actinomycin D, vincristine or adriamycin. They exhibited distinct quantitative differences of cross-resistance profiles, and showed amplification and marked expression of the mdrl/P-glycoprotein gene. DNA and RNA were prepared from the cell lines, and additionally from three cell samples of patients suffering from acute lymphatic leukemia. Applying the polymerase chain reaction (PCR) for amplification, we cloned and sequenced from these sources segments of the mdrl/P-glycoprotein gene around the codon 185 which codes for an amino acid residue possibly influencing the drug binding function of the P-glycoprotein. Altogether, only 2 single nucleotide differences in an intron were found in 2 out of 40 recombinants each harboring a 209 bp genomic or a 269 bp cDNA fragment of the mdrl/P-glycoprotein gene. Our result does not support the idea of clustered point mutations in this segment of the P-glycoprotein gene as a cause of different multidrug resistance profiles. We additionally examined another segment of the P-glycoprotein gene in its second half, essentially delivering the same negative result, though.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Differential regulation of HLA class I genes by interferon.

Allele-specific differences in the regulation of HLA class I genes by type I interferon (IFN) were observed after transfection of eight HLA-B, -A, or -C genes into mouse L cells. HLA-B7 and -Bw64 gene expression was significantly more inducible by type I IFN than the genes coding for HLA-B27, HLA-B51, HLA-B38, HLA-B39, HLA-Cw3, and HLA-A2 antigens. Modification of the 5' end of HLA-B7 and HLA-B27 genes revealed the presence of enhancer sequences responding to IFN treatment in the 5' untranslated region of HLA-B7, but not of HLA-B27 and suggested further, independently acting enhancer elements downstream of the transcription initiation site. Comparison of 5' enhancer region sequences in correlation with type I IFN inducibility of the different HLA class I alleles indicated that the exchange of only two nucleotides in the interferon response sequence (IRS) or enhancer A region of HLA-B7 or -Bw64 could account for nonregulated promoters in all other HLA-A, -B, or -C alleles analyzed. Thus, type I IFN stimulation of HLA class I genes in mouse L cells appears to predominantly operate in most alleles by a mechanism targeted to enhancer sequences downstream of the gene's transcription initiation site.

Alleles↗
PubMed1979

Isolation of replication forks from growing Ehrlich ascites cells.

A procedure is described which permits the large-scale isolation of essentially complete replications forks from the DNA of Ehrlich ascites cells. The whole nuclear DNA is first isolated by a method which involves minimal hydrodynamic shear. The DNA is then degraded by cryolysis, a freeze-thawing procedure, to a size providing the otherwise very labile forked structures with a sufficient resistance against shear forces. Finally, the Y-shaped structures of replicating DNA are separated by nitrocellulose column chromatography. When the newly formed strands of replicating DNA were density-labeled with 5-bromodeoxyuridine the DNA fraction isolated by this procedure banded in isopycnic CsCl gradients at a density expected for Y-shaped molecules with two light-heavy branches and one light-light branch and sedimented significantly faster than the corresponding bulk DNA fraction through neutral sucrose gradients. The forked molecules could be visualized by electron microscopy. The essential step of the procedure is the cryolysis which produces fragments from larger DNA structure essentially at random. When the cryolysis is omitted the forked structures are disrupted within the highly susceptible regions around the branching point.

Animals↗
PubMed1979

Random fragmentation of replicative DNA structures of eucaryotes.

The expectation values of the sizes of the different types of fragments containing replication forks obtained by random fragmentation of the DNA of growing mammalian cells have been derived. Fragments containing a single fork are expected to have, on the average, three branches of the length of a mean linear fragment thus exceeding the molecular weight of the latter by a factor 3. This factor is about 2 for fragments containing two forks very recently started from a common initiation point, and about 4 for fragments containing two forks meeting each other at the end of adjacent replication units. Only forks very close to one another are expected in these latter fragment types. Therefore, they should represent the earliest or the latest stages of replicon action.

DNA↗
PubMed1978

Differences in the binding of methylated albumin to non-replicating, replicating and denatured DNA from Ehrlich ascites cells.

The binding of methylated albumin to DNA, the basis of the chromatography on columns of kieselguhr coated with methylated albumin (MAK chromatography), was investigated. Scatchard plots revealed only one mode of interaction with fully double-stranded DNA. The complexes should be completely dissociated by raising the NaCl concentration of the solution to 0.8 M, indicating a binding by electrostatic attraction between the oppositely charged protein and DNA molecules. In complexes with denatured and partially single-stranded replicating DNA an additional kind of binding was found which made these complexes more stable against salt dissociation. These secondary interactions were stronger at 23 degrees C than at 0 degrees C and could be weakened by the addition of 6 M urea. It was therefore concluded that apolar forces were involved in these interactions.

Animals↗
PubMed1976

Newly synthesized mammalian cell DNA. Separation of Okazaki pieces by thermal chromatography on hydroxyapatite.

The optical melting profile of a fraction of nascent DNA which can be isolated from DNA of logarithmically growing Ehrlich ascites cells by nitrocellullose column chromatography was recorded. In relation to the bulk DNA a slightly lowered Tm and an increased width of the transition was observed. Thermal hydroxyapatite chromatography revealed that melting of the nascent DNA begins at relatively low temperatures in helix regions containing small daughter strand pieces. The pieces were characterized as Okazaki pieces by determining their size and their labeling kinetics during [3H] thymidine pulses. The combination of nitrocellulose chromatography and thermal hydroxyapatite chromatography seems to be suitable for isolating Okazaki pieces.

Animals↗
PubMed1975

[Repair replication in synchronous Ehrlich ascites cells after reversal of a methotrexate block (author's transl)].

If synchronous Ehrlich ascites cells are arrested at the beginning of the S phase by a methotrexate block, and the block is reversed by addition of thymidine to the culture, the cells incorporate a considerable amount of this thymidine into parental DNA within the first minute. These newly synthesized segments are irregularly distributed among the total parental DNA. This kind of distribution is expected if these segments are the result of excision repair. On the basis of the kinetics of the process this repair probably is DNA synthesis in regions already excised during the methotrexate block.

Animals↗
PubMed1975

[The secondary structure and nitrocellulose affinity of freshly replicated DNA from Ehrlich ascites cells (author's transl)].

Hydroxyapatite chromatography and isopycnic Cs2SO4 centrifugation normally yield no indications of single-stranded DNA when that fraction of replicating DNA from Ehrlich ascites cells which can be separated by nitrocellulose chromatography is analyzed. Single-stranded DNA is detected by both methods if the DNA is fragmented by ultrasound before the nitrocellulose chromatography. The digestion of this DNA fraction by single-strand-specific nucliase leads to a loss of its binding to nitrocellulose and of the indications of single-stranded DNA. The loss for the affinity to nitrocellulose is also observed when the corresponding fraction separated from unfragmented DNA is digested by endonuclease. It is suggested that replicating DNA is bound to nitrocellulose by means of single-stranded gaps on the replication fork. These gaps are apparently too small to be detected within large, otherwise entirely double-stranded molecules by hydroxyapatite chromatography and Cs2SO4 centrifugation. In the case of nitrocellulose-binding ultrasound fragments, this relation seems to be more favorable because of the separation of most of the residual double-stranded part. It is demonstrated that sonication of helical DNA also generates a small amount of fragments with some single-stranded character. The effects observed with replicating DNA could be distinguished from these artifacts.

Animals↗
PubMedDate not supplied

MDR hamster cells exhibiting multiple altered gene expression: effects of dexniguldipine-HCl (B859-35), cyclosporin A and buthionine sulfoximine.

An actinomycin D selected, multidrug-resistant (MDR) hamster CHO subline showed strong expression of the P-glycoprotein and sorcin genes together with several other alterations such as a: (i) reduced growth rate, (ii) lowered topoisomerase II, (iii) lowered glutathione-S-transferase-P gene expression, and (iv) the emergence of a 15.5 kDa protein. Besides high resistances to adriamycin, actinomycin D, and vincristine, we observed a lowered sensitivity towards bleomycin, a rather hydrophilic drug usually not involved in P-glycoprotein associated MDR. Moreover, the MDR subline showed a pronounced collateral (enhanced) sensitivity towards the sterically pure dihydropyridine anticancer drug dexniguldipine-HCl (B859-35) preventing its characterization for MDR modulation here. At a non-cytotoxic dose (10 microM) the immunosuppressive cyclic peptide cyclosporin A completely abolished the resistance to vincristine, partially reversed the resistance to teniposide and strongly enhanced the sensitivity towards bleomycin, while not influencing the drug sensitivities of the parental cell line. Buthionine sulfoximine (BSO), an agent depleting cellular glutathione levels, distinctly increased the sensitivity towards teniposide at nontoxic doses (50 microM) exclusively in the MDR subline, while it did not alter vincristine or bleomycin cytotoxicity.

Adenine Phosphoribosyltransferase↗