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R Chalkley

Publications and source records attributed to R Chalkley.

At least 73 records · Page 4Linked to original sources
PubMed1981

A new method for the isolation of replicative chromatin: selective deposition of histone on both new and old DNA.

We have developed a new method for isolating subcellular components after fixation of whole cells with formaldehyde. By a number of criteria we establish that the fixation does not alter or cause rearrangement of nucleosomal structure of either newly replicated or old chromatin. Using this approach we can almost completely resolve newly replicated chromatin from preexisting material on the basis of the difference in density. Newly replicated chromatin (even from cycloheximide-treated cells) appears to contain nucleosomes on both daughter strands. Exploiting the ability to separate newly synthesized chromatin, we have reexamined the question of the deposition of new histone at the replication fork. We find that newly synthesized histones H3 and H4 are deposited onto new DNA and stay in place for a significant time. In contrast new H1 is deposited on old DNA and new H2A-H2B, while they may be transiently bound to new DNA, are largely associated with preexisting chromatin.

Animals↗
PubMed1981

Use of whole-cell fixation to visualize replicating and maturing simian virus 40: identification of new viral gene product.

Formaldehyde fixation of simian virus 40 (SV40)-infected CV-1 cells at appropriate times after infection permits us to isolate crosslinked complexes of SV40 minichromosomes during the time of DNA replication and during packaging with viral proteins. Such crosslinked complexes can be separated on the basis of density on CsCl/guanidine . HCl density gradients. During the course of these studies we observed the presence of a low molecular weight protein in a region of the gradient much enriched with viral nucleoproteins. This protein is present only in infected cells and has a molecular weight and amino acid composition consistent with it being the product of the so-called SV40 agnogene.

Amino Acid Sequence↗
PubMed1980

H3-specific nucleohistone kinase of bovine thymus chromatin. Purification, characterization, and specificity for threonine residue 3.

Bovine thymus chromatin contains a cAMP-independent histone kinase which is entirely specific for a single site on H3 whether the histone substrate is soluble or associated with DNA in chromatin (Shoemaker, C. B., and Chalkley, R. (1978) J. Biol. Chem. 253, 5802--5807). The H3-kinase has been extracted, purified 2000-fold and extensively characterized. The purified enzyme produces a single band upon neutral gel electrophoresis and two distinct bands of 21,000 and 23,000 daltons upon sodium dodecyl sulfate-gel electrophoresis. Following subcellular fractionation, most or all of the enzymatic activity is associated with chromatin. Soluble histone inactivates H3-kinase after short incubations while a chromatin substrate permits the enzyme to remain active until H3 is fully phosphorylated. Assay conditions have been optimized in terms of pH and several cofactor concentrations. Optimal MgCl2 concentration occurs at 50 mM, while for MnCl2 the optimum is 300 microM. H3-kinase has a molecular weight of 38,000 as estimated by exclusion chromatography. The Km for ATP is 160 +/- 23 microM. The enzyme displays extraordinary substrate specificity for H3 histone as no other thymus protein has been observed as a substrate. Phosphorylation of H3 occurs at threonine residue 3.

Animals↗
PubMed1980

The identification of distinct populations of acetylated histone.

We have utilized sodium butyrate to inhibit histone deacetylases in order to study the rates of histone acetylation in hepatoma tissue culture cells. In this manner, we have been able to observe two rates of hypermodification of acetylated core histone. By selectively radolabeling acetylated histone fractions based upon differences in their acetate exchange rates, we have identified the rate of histone acetate hydrolysis and the rate of hyperacetylation in 50 mM sodium butyrate for two distinct populations of acetylated histone. One population, comprising no more than 15% of each of the non-H1 histones, is characterized by rapid hyperacetylation (t 1/2 congruent to 7 min for monoacetylated H4) and the rapid (t 1/2 congruent to 3 to 7 min) removal of this modification. A second population is deacetylated with t 1/2 congruent to 30 min and is hypermodified much less vigorously in 50 mM sodium butyrate (t 1/2 congruent 200 to 300 min for monoacetylated H4). Unlike the rapidly metabolized group, the fraction of total histone in this slow population varies between the four core histones. In addition, there appears to be no interconversion of histone between these populations.

Acetates↗
PubMed1980

Segregation of rapidly acetylated histones into a chromatin fraction released from intact nuclei by the action of micrococcal nuclease.

It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.

Acetates↗
PubMed1979

Histone deacetylation in nuclei isolated from hepatoma tissue culture cells. Inhibition by sodium butyrate.

Nuclei from hepatoma tissue culture (HTC) cells were isolated by standard methods and incubated in media commonly used for nuclease digestions (DNAase I and micrococcal nuclease) and for in vitro RNA synthesis. During the incubation, histones can be deacetylated from both control cells and cells treated with 6 mM sodium butyrate to enhance the levels of histone acetylation. Deacetylation of histone is much more apparent in nuclei isolated from sodium butyrate-treated cells. Inclusion of 6 mM sodium butyrate in the incubation medium effectively inhibits the endogenous deacetylase activity acting on histones H3 and H4, whereas sodium acetate at the same concentration has very little inhibitory effect.

Acetates↗
PubMed1979

Comparative studies on highly metabolically active histone acetylation.

Histone acetate is hydrolyzed rapidly in logarithmically dividing hepatoma tissue culture cells (Jackson, V., Shires, A., Chalkley, R. and Granner, D.K. (1975) J. Biol. Chem. 250, 4856--4863). The phenomenon has been analyzed further in hepatoma tissue culture cells at various stages of the cell cycle, in stationary phase, and in the presence of actinomycin D. We also investigated the phenomenon in Tetrahymena pyriformis macronuclei, bovine thymocytes, and human foreskin fibroblasts. The data suggest that this highly metabolically active histone acetylation while altered in mitotic cells, is independent of the overall rate of cell division, and is only slightly sensitive to actinomycin D. Finally, we conclude that the same general phenomenon is found in both cancerous and normal cells and is apparently common to cells from various stages of the evolutionary scale.

Acetylation↗
PubMed1979

Nuclease digestion studies of mouse chromatin as a function of age.

Chromatin is organized into a repeating structure (nucleosome) made up of proteins and DNA. Micrococcal nuclease and DNAase I have been used to probe this structure in nuclear populations from three tissues (liver, brain and heart) of the inbred mouse strain C57BL at different ages. For those parameters examined in each tissue, chromatin contained essentially the same features of nucleosomal organization, regardless of the age of the mouse. Thus, the rate and extent of nuclease digestion, the size of the DNA repeat unit and nucleosome core are not significantly different as a function of age. However, the accessibility of internucleosomal DNA to micrococcal nuclease, as determined by measuring the DNA size distribution after nuclease cutting, may be partially limited in brain chromatin (but not liver or heart) of older animals. These results indicate that there are no gross, age-related changes in the conformational state or organization of chromatin in these tissues. The results do not exclude smaller alterations in chromatin which might occur with age and which the methodology employed might not be sensitive enough to detect.

Aging↗
PubMed1979

A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation.

Hepatoma tissue culture (HTC) cell nuclei were digested with either DNase I or micrococcal nuclease and the nucleohistone digestion products fractionated by gel electrophoresis or exclusion chromatography. Under appropriate conditions, gel electrophoresis demonstrates that for both nucleases, only cleavages within the nucleosome spacer regions and not within the nucleosome core lead to freely migrating nucleohistone particles. These particles consist of nucleosome cores, nucleosomes and nucleosome oligomers. Following DNase I digestion and fractionation by exclusion chromatography, analysis of the histones indicates a direct relationship between increased spacer region susceptibility to nuclease and increased nucleosomal histone acetylation. Evidently digestion sites outside the regions of DNA protected by core histones can reflect the degree of acetylation of core histones. Such a relationship is not found when micrococcal nuclease is used to digest the samples.

Acetylation↗
PubMed1979

The structural organization of mouse chromatin as a function of age.

Chromatin is organized into a repeating structure (nucleosome) made up of proteins and DNA. Micrococcal nuclease and DNAase I have been used to probe this structure in nuclear populations from three tissues (liver, brain, and heart) of the inbred mouse strain C57BL at different ages. For those parameters examined, for each tissue, chromatin contained essentially the same features of nucleosomal organization, regardless of the age of the mouse. Thus, the rate and extent of nuclease digestion and the size of the DNA repeat unit and nucleosome core are not significantly different as a function of age. However, the accessibility of internucleosomal DNA to micrococcal nuclease, as determined by measuring the DNA size distribution after nuclease cutting, may be partially limited in chromatin of brain (but not liver or heart) of older animals. These results indicate that there are no gross, age-related changes in the conformational state or organization of chromatin in these tissues. The results do not exclude smaller alterations in chromatin that might occur with age, which the current methodology might not be sensitive enough to detect.

Aging↗
PubMed1978

Effect of inhibition of DNA synthesis on histone synthesis and deposition.

We have reinvestigated the degree of coupling between DNA and histone synthesis in mammalian cells. In at least one cell line (HTC cells), the coupling is not nearly as tight as had previously been inferred from experiments with HeLa cells. The site of deposition of such histones which continue to be made in the presence of sufficient hydroxyurea to depress DNA synthesis almost totally has been studied. Deposition seems to be on material which absorbs at 260 nm. This material is not a part of the bulk chromatin and binds histone in a relatively tight manner. The possible role of such a material in histone synthesis and deposition is discussed.

Cell Line↗
PubMed1978

An H3 histone-specific kinase isolated from bovine thymus chromatin.

A substantial portion of the histone phosphorylating activity of bovine thymus chromatin can be isolated by extraction in 0.2 M NaCl. The specificity of this extract for either free histones or washed chromatin substrates was compared. The salt-extracted kinase enzymes favor H2b as the major acceptor when whole free histone is the substrate and H3 when the substrate is intact chromatin. The H3 kinase activity of bovine thymus tissue has been purified free from other detectable histone kinase activities by ammonium sulfate fractionation and is highly specific for H3 histone when assayed either with chromatin or isolated whole histone. The activity is cAMP-independent. Tryptic peptide mapping of the labeled H3 histone reveals a single site of phosphorylation. This site appears to be identical with the major site of metaphase-associated H3 phosphorylation in hepatoma tissue culture cells. No corresponding H3 phosphorylation has been detected in thymus tissue in vivo.

Adenosine Triphosphatases↗
PubMed1978

The effect of sodium butyrate on histone modification.

The hyperacetylation of histones due to treatment of cultured cells with sodium butyrate has been studied. The hyperacetylation is due to inhibition of histone deacetylase. Other short chain fatty acids including acetic, isobutyric and propionic acid also produce increased modification. Histone H4 already deposited on the chromosome can be rapidly acetylated to the extent of about 70%. That 80% of histone H4 is acetylated after a 24 hr exposure to butyrate is due to the fact that incoming H4 histone is 100% acetylated and does not return to the parental unmodified form in the presence of butyrate.

Acetates↗