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Biomedical subjects

R Knippers

Publications and source records attributed to R Knippers.

At least 127 records · Page 7Linked to original sources
PubMed1980

Chromatin-associated protein kinases specific for acidic proteins.

Protein kinases (EC 2.7.1.37) were eluted by 0.4 NaCl from chromatin of several mammalian cell typs. The enzymes were partially purified by ion-exchange chromatography, DNA-cellulose columns and sucrose gradient centrifugation. At least five different enzymes could be distinguished by their biochemical properties and their substrate specificities. Three of the enzyme activities tested phosphorylate different sets of histones, while two enzymes phosphorylate acidic nonhistone chromatin proteins or artificial substrates like casein and phosvitin. The two nonhistone protein kinases have slightly different pH and salt optima. They sediment through sucrose gradients with approx. 4 S and approx. 8 S, respectively. These enzymes are further characterized by their different substrate specificity, since they phosphorylate different, though partially overlapping sets of nonhistone chromatin proteins. Enzymes with these properties were deteced in chromatin from mouse ascites cells, bovine lymphocytes, African green monkey kidney cells and a human SV40 transformed cell line.

Animals↗
PubMed1980

Acetylation of nucleosomal histones in vitro.

A new histone-specific acetyltransferase, which is closely associated with nucleosomes prepared from lymphocyte nuclei by treatment with micrococcal nuclease, is described. The acetylating enzyme transfers [3H]acetyl groups from [3H]acetyl-coenzyme A to the endogenous histones H2A, H2B, H3 and H4 in nucleosomes as well as to free histones added to the reaction mixture. Histone H1 is not acetylated by this enzyme. The acetyltransferase was partially purified by DEAE-Sephadex and DNA-cellulose chromatography. The nucleosome-associated enzyme binds to DNA cellulose at low salt concentrations (DNA-binding acetyltransferase), while the previously described histone-specific acetyltransferases have no affinity to DNA under these conditions. This high affinity for DNA may explain the association of DNA-binding acetyltransferase with nucleosomes.

Acetyl Coenzyme A↗
PubMed1980

A histone-specific acetyltransferase is associated with simian-virus-40 chromatin.

[14C]Thymidine-labeled simian virus 40 nucleoprotein complexes were prepared from nuclei of lytically infected African green monkey kidney cells. The nucleoprotein complexes were further purified by sucrose gradient centrifugation and then incubated in the presence of [3H]acetyl-coenzyme A. We observed a transfer of [3H]acetyl groups to the endogenous histones H2B, H3 and H4. The enzyme was solubilized in the presence of 0.5 M NaCl and showed properties described for the DNA-binding acetyltransferase (J. Böhm, E. J. Schlaeger and R. Knippers, preceding paper in this journal).

Acetylation↗
PubMed1979

DNA-histone interaction in the vicinity of replication points.

Chromatin replication was studied in isolated nuclei from Concanavalin A activated lymphocytes. Digestion with micrococcal nuclease revealed that the resistant fraction of in vitro replicated DNA is associated with nucleosomes. Earlier experiments had shown that the nuclease resistant fraction of nascent DNA is composed of fragments which are shorter than the nuclease resistant fragments of bulk DNA. In this communication we demonstrate that the short fragments of nascent DNA are differently bound to nucleosome like structures compared to bulk DNA. At 0.5 M NaCl a fraction of pulse labeled labeled DNA is released from these structures and appears as free double stranded DNA of about 140 base pair length (5S DNA) while the 185 pair fragments of mature replicated DNA remain attached to nucleosomes under these conditions. The experiments may indicate that the interaction of a fraction of replicating DNA with histones differs from that of bulk DNA.

Animals↗
PubMed1978

Binding of phosphorylated histone H1 to DNA.

A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of 1 mole of histone H1. The DNA binding properties of this in vitro phosphorylated H1 were compared with those of unmodified H1. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 20mM-40mM NaCl by phosphorylated H1 than by unmodified H1. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified H1 are bound to DNA. It is therefore concluded that phosphorylated H1 binds distributively to many or all DNA molecules available (depending on the histone/DNA ratio) while unmodified H1 binds cooperatively to a fraction of the DNA molecules in the reaction mixture.

Animals↗
PubMed1978

Association of DNA polymerase with nucleosomes from mammalian cell chromatin.

More than half of the DNA polymerase beta in mouse ascites cell chromatin was found to be associated with monomeric nucleosomal particles (produced by micrococcal nuclease treatment of chromatin). Almost all nuclear DNA polymerase activity in lymphocytes was found to be associated with nucleosomes. The nucleosome-associated enzyme was mainly DNA polymerase beta in chromatin from resting and mainly DNA polymerase alpha in chromatin from concanavalin-A-stimulated lymphocytes.

Animals↗
PubMed1977

Studies on protein-phosphorylation reactions in isolated chromatin.

The endogenous protein-phosphorylating activity of isolated chromatin was tested. We have found that a group of high-molecular-weight proteins (Mr greater than 50 000) was preferentially phosphorylated when chromatin from mouse ascites cells or from bovine lymphocytes was incubated in the presence of ATP. After disintegration of chromatin by nuclease treatment or by high salt concentration, a larger spectrum of chromatin proteins becomes accessible for phosphorylation by the chromatin-bound protein kinase. Some observations described in this communication may help to partially explain this result. The protein kinase was not found in nucleosomal subunits, indicating a non-random distribution of the enzyme in chromatin. This suggests that enzyme and substrate have to be in close spatial contact for the phosphorylation reaction to occur. Furthermore, we have shown for one protein, histone H1, that phosphorylation sites for the endogenous protein kinase are available on the free but not on the DNA-bound protein, suggesting that phosphate-accepting sites in chromatin proteins may be blocked by protein-DNA or by protein-protein interactions. We also discuss the possibility that chromatin protein kinase occurs in stable complexes with its phosphate-accepting substrates, as has been suggested by the findings of other [Kish, V.M. & Kleinsmith, L.J. (1974) J. Biol. Chem. 249, 750-760].

Animals↗
PubMed1977

A single-strand-specific DNA-binding protein from mouse cells that stimulates DNA polymerase.

A protein of 30000-35000 molecular weight was isolated from mouse ascites cells. This protein binds preferentially to single-stranded DNA. Evidence is presented that this protein maintains single-stranded DNA in an extended configuration. In the presence of single-stranded template the protein stimulates mammalian DNA polymerase alpha but not the mammalian DNA polymerase beta and not some microbial DNA polymerases. The protein is phosphorylated in vitro by a chromatin-associated protein kinase. The modified DNA-binding protein does not stimulate the DNA polymerase alpha.

Animals↗
PubMed1977

[Regulation of DNA replication (author's transl)].

Two aspects of the regulatory mechanisms of DNA replication are discussed: 1. When resting mammalian cells are stimulated to proliferate a genetic program is expressed which provides proteins and other replication factors; 2. the interaction of proteins with specific sections on the DNA leads to initiation; most probably, structural changes in chromatin are also involved. Once initiated, replication proceeds almost automatically through the elongation phase. Lymphocytes stimulated to proliferate by concanavalin A are used as an example to demonstrate the relative importance of the two aspects of DNA replication.

Animals↗
PubMed1976

An endonuclease from mouse cells specific for single-stranded DNA.

An endonuclease with a molecular weight of about 70000 (5-6S) was extensively purified from mouse ascites cells. The enzyme specifically attacks single-stranded DNA which is degraded mainly to oligonucleotides, with 5-10 residues. Supercoiled covalently closed circular phage DNA is converted to the linear relaxed form. The enzyme activity is highly sensitive to salt and can be stimulated by reagents lowering the dielectric constant of the buffer such as dimethylsulfoxide and glycerol.

Animals↗
PubMed1976

Recombination of bacteriophage T7 in vivo.

Most recombination following infection with T7 was found to coincide with the time of most repid DNA synthesis, at about 20 min after infection at 30 degrees in minimal medium. Recombining DNA was investigated electron microscopically. Multiply branched DNA structures were observed after infection with T7 wild type, gene 3-, gene 6- and genes 3-, 6- phage, but not after infection with T7 gene 5- phage. Evidence is presented indicating that these structures are T7 DNA molecules in the process of recombining. The detailed structures of these recombinational intermediates suggest mechanisms by which T7 DNA initiates recombination.

Coliphages↗
PubMed1975

Nuclear protein kinases from murine cells.

Three protein kinase activities are found in nuclei from three different murine cells (Ehrlich ascites cells, mouse L cells and rat glioma cells). Two of these activities are soluble, one is bound to chromatin. The soluble enzymes are similar, if not identical, to the cytoplasmic protein kinases. The chromatin-bound, adenosine-cyclic-3':5'-monophosphate-independent enzyme is not found in the cytoplasm. This enzyme is composed of one subunit with a molecular weight of 80000-90000. Some biochemical properties of this enzyme are described. A brief description of a nuclear enzyme, which dephosphorylates phosphorylated histones, is also given.

Animals↗