Search PubMed⌕ Search

Biomedical subjects

A Weissbach

Publications and source records attributed to A Weissbach.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1984

DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides.

A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.

Cell Nucleus↗
PubMed1984

Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence.

This study presents the first evidence that the 5' promoter region of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (G-3-PD) promoter will permit expression of an adjacent foreign gene. The S. cerevisiae G-3-PD promoter was linked to the herpes simplex virus--thymidine kinase (HSV-TK) gene in a shuttle plasmid capable of autonomous replication in both yeast and Escherichia coli. Since the HSV-TK gene promoter is not functional in yeast, yeast cells containing these plasmids will express the HSV-TK gene and synthesize thymidine kinase only if the yeast promoter fragment is fused to the HSV-TK gene in the proper orientation. The 5' flanking sequences necessary for the expression of heterologous eukaryotic genes in S. cerevisiae are discussed.

Base Sequence↗
PubMed1984

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.

Cell Compartmentation↗
PubMed1983

Characterization of a prokaryotic topoisomerase I activity in chloroplast extracts from spinach.

A topoisomerase I activity has been partially purified from crude extracts of spinach chloroplasts. This activity relaxes the supercoiled covalently closed circular DNA of pBR322. The enzyme requires Mg++, but not ATP, and has an apparent molecular weight of about 115,000. It catalyzes a unit change in the linkage number of supercoiled DNA but cannot relax positive supercoiled DNA. These characteristics of the topoisomerase suggest it is of the prokaryotic type and would tend to support the endosymbiotic theory of plastid origin and evolution.

Chloroplasts↗
PubMed1982

Deoxyribonucleic acid synthesis in isolated chloroplasts and chloroplast extracts of maize.

Isolated chloroplasts are capable of synthesizing chloroplast DNA in the presence of Mg2+ and deoxynucleoside triphosphates. The in vitro reaction proceeds for at least 60 min and is inhibited by KC1 and N-ethylmaleimide. Stretches of several hundred nucleotides in length are synthesized within an hour. Little or no inhibition is shown by aphidicolin (an inhibitor of eukaryotic DNA polymerase alpha), dideoxythymidine triphosphate (an inhibitor of eukaryotic DNA polymerases beta and gamma), nalidixic acid, or rifampicin. Ethidium bromide is a moderate inhibitor of DNA synthesis in the isolated chloroplast. Soluble extracts of chloroplasts will copy exogenously added recombinant plasmid circular DNA containing fragments of chloroplast DNA, and this reaction is strongly inhibited by ethidium bromide. Copying of the plasmid DNA takes place on the relaxed circular or linear forms of the DNA, but no specific initiation sites on the chloroplasts' DNA fragments of the recombinant plasmids have been detected. Our data are consistent with a repair mechanism operating in vitro but may also represent incomplete replicative DNA synthesis.

Aphidicolin↗
PubMed1982

The isolation and characterization of DNA polymerase alpha from spinach.

A DNA polymerase with the characteristics of the alpha class of eukaryotic DNA polymerases has been purified 1000-fold from spinach leaves. The enzyme has a molecular weight of 160,000 +/- 10,000 in its native form and is markedly inhibited by aphidicolin and N-ethylmaleimide, but not by dideoxynucleoside triphosphates. As isolated, the enzyme contains no detectable deoxyribonuclease activity. A catalytically active 12-kilodalton fragment of the DNA polymerase, apparently generated by endogenous proteolytic action, has also been purified. The native enzyme is found predominantly in the cytoplasmic fraction of broken leaf cell preparations and less than 10% is found associated with the nuclei.

Cell Nucleus↗
PubMed1981

Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis.

Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.

Animals↗
PubMed1980

Mitochondrial DNA replication does not involve DNA polymerase alpha.

Aphidicolin, a known inhibitor of DNA polymerase alpha, is a potent inhibitor of nuclear DNA synthesis in HeLa cells but has no effect on the replication of mitochondrial DNA. Parallel experiments with mitochondria incubated in vitro also show no inhibition of DNA synthesis by aphidicolin; however, DNA synthesis in these isolated mitochondria is completely blocked by dideoxycytidine triphosphate, which inhibits DNA polymerase gamma but not the alpha polymerase. The replication of mitochondrial DNA therefore requires only one DNA polymerase of the gamma type.

Aphidicolin↗
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↗
PubMed1978

Initiation of HeLa cell DNA synthesis in a subnuclear system.

Mammalian cells are known to synthesize DNA in discrete stages, the first of which seems to be the formation of DNA pieces 150--200 nucleotides in length that have a s20 value of about 4 S. We have reconstructed a system derived from HeLa cell nuclei that carries out RNA-primed initiation of the synthesis of small (4S) DNA fragments. This synthesis is resistant to high concentrations of alpha-amanitin and sensitive to antibody directed against RNA polymerase I, suggesting that this enzyme may be involved in the initiation step. The formation of small DNA fragments in this system also requires DNA polymerase alpha, heat-labile nuclear factor(s), and at least one other nuclear protein.

Cell Nucleus↗
PubMed1978

Synthesis of viral and host DNA in isolated chromatin from herpes simplex virus-infected HeLa cells.

DNA synthesis in chromatin isolated from herpes simplex virus type 1-infected HeLa cells (HSV chromatin) was examined in vitro. The HSV chromatin was found to carry out an initial limited synthesis of DNA in vitro, 50 to 64 pmol of dTMP incorporated in 10(6) nuclei per 10 min, which is comparable to that found in nuclei isolated from HSV-infected cells. DNA synthesis in vitro proceeded for only 30 min, and both HSV DNA and host DNA were synthesized in significant amounts. The HSV and host DNA synthesis in isolated chromatin were inhibited to the same extent by anti-HSV antiserum or by phosphonoacetic acid. The results indicate that the HSV-induced DNA polymerase is most likely involved in the synthesis of host and HSV DNA in isolated chromatin, even though this chromatin contains small amounts of the host gamma-polymerase in addition to the HSV-induced DNA polymerase. The HSV chromatin contains no detectable levels of DNA polymerases alpha and beta, even though infected cells have normal, or increased, levels of these enzymes.

Centrifugation, Density Gradient↗