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R Chalkley

Publications and source records attributed to R Chalkley.

At least 91 records · Page 5Linked to original sources
PubMed1978

DNA associated with hyperacetylated histone is preferentially digested by DNase I.

Butyrate-treated cells give rise to massive hyperacetylation of histones and have been used to test the idea that regions of DNA in association with hyperacetylated histones are preferentially solubilized upon digestion with DNase I. Such hyperacetylated histones can be derived from both pre-existing histones or from histone newly synthesized in the presence of butyrate which leads to extreme modification. The DNA in association with both types of hypermodified histone is equally and selectively digested.

Acetylation↗
PubMed1977

Histone neighbors in nuclei and extended chromatin.

Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics and intimate histone associations in compact chromatin.

Cell Nucleus↗
PubMed1977

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.

Animals↗
PubMed1976

Production of high levels of phosphorylated F1 histone by zinc chloride.

Methods have been sought to perturb the level of phosphohistones. ZnCl2 (10 mM) exhibits histone phosphate phosphatase in vivo in HTC cells and leads to hyperphysiological levels of F1 phosphohistone. Treatment of tissue culture cells with this concentration of ZnCl2 leads to a reduction in medium pH to 6.4. Control experiments have indicated that HTC cells grow efficiently at this pH and that the reduction of pH does not produce the hyperphosphorylated state per se. The optimum conditions for the ZnCl2 effect are described. That the effect of ZnCl2 on the heterogeneity of F1 histone is due to an effect on phosphorylation was demonstrated by the observation that the entire effect is abolished by treatment with alkaline phosphatase. The site of phosphorylation is in the carboxy-terminal end of the F1 molecule. The inhibitory effect of ZnCl2 on F3 phosphorylation in metaphase cells is also described.

Alkaline Phosphatase↗
PubMed1976

Deposition of histone onto the replicating chromosome: newly synthesized histone is not found near the replication fork.

We have studied the site of deposition of newly synthesized histone. It appears to be randomly distributed over the chromosomal material and does not become associated specifically with immediately post-replicational DNA, nor is it deposited in discrete continuous regions distal to the sites of DNA synthesis. The newly synthesized DNA, however, rapidly acquires a complement of chromosomal proteins; presumably, preexisting histones must migrate to become associated with post-replicational DNA.

Cells, Cultured↗
PubMed1976

Relationship between chromosome condensation and metaphase lysine-rich histone phosphorylation.

Treatment of metaphase HTC cells with ZnCl2 inhibits histone phosphatase activity and leads to an increase in the hyperphosphorylated forms of the lysine-rich (F1) histone. Under normal conditions a massive phosphatase activity is triggered as the cells shift from M into G1 phase. In the presence of ZnCl2 this activity is abolished and thehyperphosphorylated form of F1 persists intact into G1. We have asked the simple question of whether the chromosome can still extend during the M-G1 transition even if the F1 histone is maintained in the hyperphosphorylated form. We observe an apparently normal extension os the chromosomal material under these conditions, though it is evident that high levels of ZnCl2 have rather substantial effects on other cell functions.

Cell Division↗
PubMed1975

Studies on highly metabolically active acetylation and phosphorylation of histones.

The capacity to effectively label tumor cell hostones using very short pulses of [3-H]acetate and [32-P]phosphate (1 to 10 min) has been developed. Four histone fractions F3, F2a1, F2a2, and F2b are extensively acetylated in short time periods. About 70% of the acetate accumulated on the histone during a short pulse is removed with a half-life of similar to 3 min. The rest of the metabolically active acetate is removed with a half-life of 30 to 40 min. Histones F2a1, F2a2, and F1 are acetylated at the NH2 terminus and this modification is metabolically stable. In short pulses, histones are labeled with 32-P in the order F2a2 greater than F1 greater than F3 greater than F2a1 greater than F2b. All fractions have a fairly rapid turnover time (t1/2 similar 20 to 40 min) except F1 phosphate which turns over some 5 times more slowly.

Acetates↗
PubMed1975

The phosphorylation region of lysine-rich histone in dividing cells.

N-Bromosuccinimide cleavage of in vivo 32P-labelled lysine-rich histone isolated from rapidly dividing cells has been studied. N-Bromosuccinimide cleaves F1-histone into two fragments, a small N-terminal piece and a larger C-terminal portion. The phosphate-induced microheterogeneity and associated radioactivity which has been linked to cell replication, is found in the carboxyterminal fragment, No phosphorous is found associated with the amino-terminal fragment when histone phosphorylation is associated with cell division. The specific tryptic phosphopeptides obtained from in vivo labelled F1 are clearly different from those obtained from in vitro incubations of free F1-histones and cytoplasmic protein kinase.

Animals↗
PubMed1975

Deposition of histones onto replicating chromosomes.

The mode of distribution of newly synthesized and pre-existing histones has been studied during the process of chromosome replication. Newly synthesized histone was labeled with [3H]lysine and newly synthesized DNA was density labeled with iododeoxyuridine. The histone was covalently linked to DNA, and radiolabeled histone was analyzed on CsCl density gradients. We have defined conditions that do not give rise to histone randomization during isolation, and also developed a method of defining the distribution of histones in chromatin on a density gradient in the unavoidable presence of nonhistone protein. Three possible modes of distribution of histone onto the replicating chromosome can be conceived; we describe experiments designed to distinguish unequivocally among these possibilities and conclude that histones are deposited randomly onto the chromosome.

Carcinoma, Hepatocellular↗