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Biomedical subjects

A Weissbach

Publications and source records attributed to A Weissbach.

At least 19 recordsLinked to original sources
PubMed1990

Genomic sequencing of the 5'-flanking region of the mouse beta-globin major gene in expressing and nonexpressing mouse cells.

Genomic sequencing of two CG sites located in the 5'-flanking promoter region of the mouse beta-globin major gene shows these sites to be heavily methylated in the DNA from L929 mouse fibroblasts, a cell line that does not express the beta-globin gene. By contrast, the same CG sites in the DNA obtained from murine erythroleukemia cells, which can express the beta-globin gene, are unmethylated. The results suggest that either differentiation of progenitor cells to form the erythroid precursor murine erythroleukemia cell and/or transformation by the Friend virus group leads to demethylation of these CG sites. Final activation of the transcription process for the mouse beta-globin gene does not require demethylation in the 5'-promoter region of the DNA.

5-Methylcytosine↗
PubMed1990

Organellar DNA replication in Nicotiana tabacum cultured cells.

In the diploid vegetative plant cell, the nuclear DNA is present in two copies, whereas the chloroplast and mitochondria genomes are present in a higher and variable copy number. We have studied the replication of the nuclear, chloroplast and mitochondrial DNA in cultured Nicotiana tabacum cells using density and radioactive markers. Essentially all the 10,000 chloroplast genomes in a given cell replicate in one cell cycle as do all the mitochondrial DNA molecules. No measurable level of unreplicated organellar DNA molecules can be detected in these cells.

Bromodeoxyuridine↗
PubMed1987

In vitro methylation of the 5'-flanking regions of the mouse beta-globin gene.

The enzymatic methylation of the 5'-flanking region of the mouse beta-globin (major) gene containing putative regulatory regions has been investigated. In vitro methylation of this 368-base pair regulatory DNA by a DNA methyltransferase obtained from mouse erythroleukemia cells yields an asymmetric methylation pattern. Of the 10 available CG pairs, only 5-6 are modified, leading to one hemimethylated site and two apparently fully methylated sites. Only CG pairs which are localized in a 29-base pair cluster are methylated. The data suggest that a CG cluster approximately 100 base pairs upstream from the CAP site may be the in vivo site of methylation in the 5'-regulator region of the mouse beta-globin gene.

Animals↗
PubMed1987

[Microinvasive stage Ia cancer of the uterine cervix--a population based analysis].

In a population based study, 782 cases of microinvasive cervical cancer stage Ia recorded in Berlin, capital of the GDR, between 1970 and 1978 were analyzed. As the consequence of a cytological screening programme started in 1973, the percentage of stage Ia-cancer increased to 30% in 1978 and 35% in 1984. The percentage of asymptomatic cases (86%) only detectable by colposcopy and/or cytology (92%) increased also considerably. The percentage of cases histologically confirmed by cone biopsy increased from 55% to 81%. During the period analyzed, the percentage of radically treated cases (Wertheim, Schauta, combined surgery and radiotherapy) decreased to 31%. Among 459 patients only treated by conization or simple hysterectomy 7 developed recurrences and 2 died from the disease despite of an intensive second treatment. Among 323 patients treated more radically (Wertheim, Schauta, combined surgery and radiotherapy), there was no recurrence but 5 deaths by early or late treatment complications. Therefore, observed cumulative survival rates of patients treated by conization or hysterectomy were slightly higher than those of patients treated more radically.

Adult↗
PubMed1986

Mammalian DNA methyltransferases prefer poly(dI-dC) as substrate.

The synthetic duplex DNA, poly(dI-dC).poly(dI-dC), is methylated in vitro by human or murine DNA methyltransferases at 20-100 times the rate of other nonmethylated DNAs. Preparation of the hemimethylated derivative, poly(dI-dMeC).poly(dI-dC), of this polymer increases its effectiveness as a substrate by 2-fold, making it 4-10 times more effective as a substrate for mammalian DNA methyltransferases than any other hemimethylated DNA so far reported. However, the apparent slower rate of de novo methylation of poly(dI-dC).poly(dI-dC) as compared to the hemimethylated derivative is due to substrate inhibition, unique to the unmethylated polymer, as the rates of de novo and maintenance methylation are identical at low substrate concentrations.

Animals↗
PubMed1986

Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases.

Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs.

Animals↗
PubMed1985

Quantitative molecular hybridization on nylon membranes.

A study of DNA hybridization to DNA covalently bound to nylon membranes was made in order to develop a quantitative method for molecular hybridization using a nylon-based matrix. Chloroplast DNA was covalently attached to nylon membranes by irradiation at 254 nm. Under hybridization conditions the initial rate of DNA loss from the nylon membranes was 5-10% per 24 h, while under comparable conditions DNA bound to nitrocellulose membranes was lost at a rate of 38 to 61% per 24 h. Several sets of hybridization conditions were examined to select one giving reasonable hybridization rates and minimal loss of bound DNA. Under the conditions selected [Denhardt's solution (D. Denhardt, 1966, Biochem. Biophys. Res. Commun. 23, 641-646), 0.5 M NaCl, 0.1% sodium dodecyl sulfate, and 31.4% formamide at 50 degrees C for 92 h], hybridization was observed to be 29% more efficient on nylon membranes than on nitrocellulose. Several attempts to remove previously hybridized DNA from nylon membranes proved only partially successful. Reuse of the membranes, therefore, was of limited value. Quantitative hybridization of total radiolabeled tobacco cellular DNA to cloned tobacco chloroplast DNA attached to nylon yielded results similar to those previously reported using nitrocellulose membranes. However, use of nylon membranes greatly facilitated the manipulations required in the procedure.

Binding Sites↗
PubMed1985

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.

Base Composition↗
PubMed1985

Chloroplast DNA synthesis during the cell cycle in cultured cells of Nicotiana tabacum: inhibition by nalidixic acid and hydroxyurea.

The effects of nalidixic acid and hydroxyurea on nuclear and chloroplast DNA formation in cultured cells of Nicotiana tabacum were investigated. At low concentrations (5 and 20 micrograms/ml) nalidixic acid, an inhibitor of DNA gyrase, exhibited a greater inhibitory effect on plastid DNA synthesis than on nuclear DNA formation. Since the plastid genome is a circular double-stranded DNA, this is consistent with the proven involvement of a DNA gyrase in the replication of closed circular duplex DNA genomes in procaryotic cells. At a high concentration of nalidixic acid (50 micrograms/ml), DNA synthesis in both the plastid and nuclear compartment was rapidly inhibited. Removal of the drug from the culture medium led to the resumption of DNA synthesis in 8 h. Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, also depresses nuclear as well as plastid DNA formation. Removal of hydroxyurea from the blocked cells leads to a burst of nuclear DNA synthesis, suggesting that the cells had been synchronized at the G1/S boundary. The recovery of plastid DNA synthesis occurs within the same time frame as that of nuclear DNA. However, whereas plastid DNA formation is then maintained at a constant rate, nuclear DNA synthesis reaches a peak and subsequently declines. These results indicate that the synthesis of plastid DNA is independent of the cell cycle events governing nuclear DNA formation in cultured plant cells.

Cell Cycle↗
PubMed1985

Enzymeimmunoassay of unconjugated estriol in serum and saliva during pregnancy.

An enzymeimmunoassay (EIA) for unconjugated estriol was developed. Estriol-6-CMO-BSA was used as antigen for the antiserum production with low cross reactivities to estrone (0.3%) and estradiol (0.17%). The enzyme conjugate (estriol-peroxidase) was prepared using the mixed anhydride reaction. The antibody bound estriol fraction was separated from the free fraction with the double antibody technique. The test requires a simple photometer for the measurement of enzyme label. The developed estriol-EIA needs a short incubation time (2 hrs). The normal values of estriol in serum were determined. The results are in good agreement with the results of RIA. Furthermore, we found an inverse diurnal rhythm of serum and salivary estriol in comparison to the cortisol level. Low estriol levels in the morning and high estriol concentration in the evening are indices of fetal health. Decreased estriol levels in the morning of several days were found in pathological pregnancies (intrauterine fetal death, fetal distress, hepatose).

Circadian Rhythm↗
PubMed1985

The effect of flanking sequences on the de novo methylation of C-G pairs by the human DNA methylase.

The HeLa DNA methylase can methylate selected cytosine residues in oligodeoxynucleotides as small as 12-16 nucleotides in length in vitro. The maximum methylation rate seems to require oligomers having more than one C-G in the molecule even when only one of the C-G pairs is methylated. Compounds which contain a high G+C content also seem to be favored substrates. The use of defined synthetic oligodeoxynucleotides permits one to demonstrate that flanking DNA sequences can be critical in determining whether a C-G site can be methylated.

Base Composition↗
PubMed1979

Persistence of herpes simplex virus type 1 in rat neurotumor cells.

Herpes simplex virus type 1 (HSV-1) infection of a rat central nervous system tumor cell line led to almost complete destruction of the cells. Cells that survived the infection could be isolated and shown to produce infectious HSV particles for variable lengths of time in culture ranging from 20 to 57 passages. Even though infectious virus production eventually ceased, the cell lines continued to produce herpes-specified proteins as measured by immunological techniques. These cells also showed herpesvirus-like structures in the electron microscope. The persistently infected cells that produced HSV antigens and bore HSV sequences were resistant to superinfection by HSV-1. The resistance was not due to failure of adsorption of the virus or to the production of interferon by the cells. The nature of the block in HSV replication in these neurotumor cells, which contain and partially express the HSV genome, is unknown, but may offer an interesting parallel to the known latency of HSV in neural tissues.

Animals↗