Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine.

The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.     

Variations in rat liver chromatin composition during growth and the heterotic response

The manifestation of hybrid vigor with regard to the inheritance of body weight was established as a postweaning phenomenon in rats. Liver weight gain generally corresponded to body weight gain and also reflected hybrid vigor. The protein/DNA ratios of the chromatin from livers of inbred and heterotic hybrid male rats were found to increase with increasing age in growing rats. Hybrid chromatin had higher protein/DNA ratios throughout the age range studies. The difference between hybrids and inbreds were greatest in the early postweaning stages of growth and maturation. The RNA/DNA ratios of chromatin from livers of inbred and hybrid male rats were also found to exhibit trends within certain developmental periods.This investigation was supported by matching Federal and West Virginia State funds from the Hatch 190 grant and is published with the approval of the Director of the Agricultural Station as Scientific Paper No. 1293.     

Effect of insulin on the composition of chromatin phospholipid in rat liver cells

In vivo the action of insulin on rat liver chromatin phospholipid composition was investigated. It was shown that the hormone led to reliable increase (nearly 20%) of total phospholipid content. The same phenomenon was shown also in the fraction of active chromatin while the phospholipids content of non-active chromatin didn't changed. It was also shown that the content of three from six phospholipid fractions altered under the insulin action. The content of sphingomyelin and phosphatidilethanolamine increased and the quantity of phosphatidylinositol decreased under the hormone action. The decrease of monophosphoinositides content was accompanied by the reliable increase of triphosphoinositides amount. It was suggested that the fractions of chromatin polyphosphoinositides were redistributed under the action of insulin.     

Virogenic BrdU and BrdU-sensitive DNA sequences are disproportionately concentrated in the template-active chromatin of rat embryo cells

In order to characterize the molecular mechanism responsible for the BrdU-mediated activation of endogenous retrovirus from normal rat embryo cells, the previously reported selective distribution of bromouracil in DNA was correlated with the corresponding organization of the nucleo-protein complex in regard to nucleosome structure and template - active and -inactive chromatin. Following micrococcal nuclease digestion of chromatin labeled with either [(3)H]thymidine or [(3)H]BrdU, the amount and specific activities of the respective nucleosomal DNA were indistinguishable. Comparable findings were obtained following direct examination of the nuclease-sensitive, "spacer" DNA. However, when each chromatin type was fractionated into template-active and -inactive components, it was evident that [(3)H]bromouracil was nonrandomly more concentrated in the template-active portion in comparison to the random distribution of [(3)H]thymine moieties. Furthermore, it was apparent that the template-active chromatin fraction was substantially enriched in the nucleotide sequences of rat DNA known to be sensitive to the virogenic action of BrdU.     

Conformational changes in rat liver chromatin after liver regeneration.

N-Pyrenemaleimide, a fluorescent probe that specifically labels histone H3 of rat liver chromatin in situ, was used to monitor the accessibility of histone H3 in chromatin isolated from rat liver at different times during degeneration. At times of maximum DNA synthesis (18--24 h after hepatectomy), the accessibility of the probe was found to be markedly (40--50%) increased. This increase is abolished, however, by treatment of the chromatin fibres with high salt (2 M-NaCl) or detergent. Tryptophan fluorescence was also enhanced at points of maximum DNA synthesis, suggesting that some non-histone tryptophan-containing protein was being synthesized. The polarization of the labelled histone H3 is not markedly altered, suggesting that fibre aggregation or dissociation does not occur. Mononucleosomes extracted from sham-operated and hepatectomized animals did not exhibit any difference in binding to the probe. Also, analysis of the chromatin protein by electrophoresis on detergent- and acid/urea/ Triton-X-100-containing polyacrylamide gels showed no detectable difference in histone H3 : 1, H3 : 2 or H3 : 3 subclasses.     

Action of hydrocortisone on the lipid composition of active and inactive chromatin fractions in the rat liver

A composition of phospholipids and neutral lipids of rat liver chromatin and its active and inactive fractions has been investigated. It is shown that the hydrocortisone action results in marked increase in phospholipid/neutral lipid ratio of both chromatin and its active fraction. The changes in lipid content is clearly expressed in active chromatin fraction, the lipid content of inactive fraction is not changed. It is concluded that the increase of content in certain phospholipids and simultaneous decrease of neutral lipids in chromatin promotes the hormonal activation of genome.     

Purification of a phosphoprotein from chromatin of rat liver.

A simple and effective method to purify a phosphoprotein (B2) (Mr 68,000, pI 6.2-8) from phenol-soluble non-histone chromatin proteins of rat liver is described. The purification involved only two steps, CM-cellulose chromatography and preparative SDS/polyacrylamide (10%)-gel electrophoresis. The purified phosphoprotein B2 was shown to be homogeneous by SDS/polyacrylamide-gel electrophoresis. The yield was 2% of total non-histone chromatin proteins. The acidic to basic amino acid ratio of phosphoprotein B2 was less than 1, with high contents of glutamic acid, aspartic acid, arginine, lysine, glycine and alanine. The phosphate content of this protein is 0.3%.     

Reverse sphingomyelin-synthase in rat liver chromatin

The chromatin phospholipid fraction is enriched in sphingomyelin content which changes during cell maturation and proliferation. Recently, we have demonstrated that the sphingomyelin variations can be due to chromatin neutral sphingomyelinase and sphingomyelin-synthase activities which differ in pH and K(m) optima from those present in nuclear membranes. The sphingomyelin can be used also as a source of phosphorylcholine for phosphatidylcholine synthesis by reverse sphingomyelin-synthase. In the present work we have studied the possible existence of reverse sphingomyelin-synthase activity in nuclear membrane and chromatin. A very low activity was detected in the homogenate, cytosol and nuclear membrane (0.93+/-0.14, 2.61+/-0.33 and 0.87+/-0.13 pmol/mg protein/min, respectively), whereas the activity present in chromatin was 37.09+/-2.05 pmol/mg protein/min. The reverse sphingomyelin-synthase decreases the intranuclear diacylglycerol pool and increases the intranuclear ceramide pool, whereas sphingomyelin-synthase has an opposite effect. The possible correlation between these enzymes is discussed.     

Effect of triiodothyronine on rat liver chromatin protein kinase.

1. Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2. Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32P when incubated with [gamma-32P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins.