CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-³H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.     

RNA Stimulated by Indole Acetic Acid

THE stimulation of RNA synthesis in plant cells by auxins^1,2 may be due, in part, to gene derepression, since chromatin isolated from hormone-treated cells is a better template. Mathysse and Phillips³ demonstrated that an acceptor protein for auxin can interact with the chromatin to derepress the genome. In their system the hormone and protein do not affect the rate of RNA synthesis if pure DNA. is used as a template. However, the actual mechanism of auxin on RNA synthesis by isolated RNA polymerase system has yet to be elucidated.     

Effects of N-methyl-N-Nitrosourethane on DNA synthesis in the guinea pig pancreas

The effects of N-methyl-N-nitrosourethane (NMUT) on pancreatic DNA synthesis were investigated at sequential intervals following gavage of Hartley guinea pigs with a single dose of 30 mg/kg. There was a highly significant stimulation of DNA synthesis, as evidenced by increased incorporation of [³H] methyl-thymidine ([³H] TdR), throughout the whole pancreas and particularly in the duodenal segment, at 4 h following NMUT administration, thereafter, DNA synthesis declined sharply up to 24 h, and then recovered gradually to control levels from 24–96 h. DNA synthesis stimulated by NMUT was suppressed by hydroxyurea (HU), and hence is likely to represent replicative, rather than repair, synthesis.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.     

Changes in chromatin of Daucus carota cells during embryogenesis

Cells of carrot (Daucus carota var. Rote Riesen) were cultured on media inductive and non-inductive for embryogenesis and analyzed for differences in their chromosomal proteins and chromatin template activity. Non-histone proteins were prepared from dehistonized chromatin and their properties were investigated. Non-histone proteins proved to be acidic and associated easily with calf thymus histone. Non-histone proteins were able to counteract the inhibitory effect of histone on DNA-directed RNA synthesis in vitro. Almost the same rate of restoration occurred regardless of the interaction between DNA and protein, when sufficient amounts of non-histone proteins were added. However, once the histone-DNA complex was established, the restoration by non-histone proteins at comparably lower concentration was poor. Another acidic protein, bovine serum albumin, had no effect on histone inhibited RNA synthesis. Also non-histone proteins enhanced the chromatin directed RNA synthesis more than 100%. The template activity of chromatin changed after the inductive treatment of embryo formation and induced cells showed higher template activity than non-indiiced controls after embryo cells were formed. Histone components were the same in inductive and non-inductive cells. On the other hand, there was a correlation between template activity and the stimulation by non-histone proteins of histone-inhibited RNA synthesis.     

Aspects of DNA, RNA and protein synthesis during somatic embryogenesis in a carrot cell suspension culture

Changes in DNA, RNA and protein content, incorporation of ³H-thymidine, ¹⁴C-uridine and ³H-leucine and template activity of chromatin were investigated in the early process of somatic embryogenesis in a carrot (Daucus carota L. cv. Kurodagosun) cell suspension culture using a synchronous system. An embryogenetic culture in a medium containing 10^-7M zeatin was compared with a non-embryogenetic culture in a medium containing 10^-7M zeatin and 5 x 10^-7M 2,4-D. DNA was synthesized very actively prior to and during the formation of globular embryos in the embryogenetic culture. The RNA and protein content per tube increased at an almost constant rate in both cultures, while the rate of incorporation of labelled precursors of RNA and protein rose much more prior to active DNA synthesis in the embryogenetic culture than in the non-embryogenetic culture. Template activity of chromatin was high in the early stage of embryogenesis in the embryogenetic culture. The results obtained here showed that synthesis and turnover of RNA and protein became active prior to active DNA synthesis in the early stage of embryogenesis, and that these changes at macromolecular levels may play important roles in embryogenesis.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

PHA stimulation of human lymphocytes during amino acid deprivation. Protein,RNA and DNA synthesis

Resting lymphocytes are in the G₀ phase of the cell cycle. Upon activation by PHA, they progress into G₁ with accompanying increased protein and RNA synthesis, initiate DNA synthesis and divide. We have studied the kinetics of inhibition of macromolecular synthesis during activation in the absence of single amino acids. Three types of kinetics are observed. In the absence of tryptophan or isoleucine, stimulated lymphocytes show a normal increase in protein and RNA synthesis during the first 30 hours of stimulation, initiate DNA synthesis but are subsequently inhibited. In phenylalanine-deficient medium, no DNA synthesis occurs in spite of a slight increase in protein synthesis. No increase in macromolecular synthesis is observed in medium lacking any one of the other essential amino acids (eg: lysine). Our results indicate that the kinetics of macromolecular synthesis in tryptophan-deficient medium is the result of a limited reserve of protein-bound tryptophan which becomes exhausted after 30 hours. On the other hand, delayed inhibition of synthesis in isoleucine-deficient medium probably reflects an initially low requirement for this amino acid followed by inhibition of the synthesis of isoleucine-rich proteins involved in some late event of stimulation. Partial deprivation of lysine results in kinetics of protein synthesis similar to that in tryptophan- or isoleucine-deficient media. The results indicate that the kinetics of macromolecular synthesis during activation of lymphocytes in the absence of an essential amino acid is a function of the quantitative requirement for that amino acid, at a given time during stimulation. Upon replacement of lysine, lymphocytes inhibited by lysine deficiency begin RNA and protein synthesis immediately and at a rate faster than that of unstimulated cultures to which PHA is added. They also initiate DNA synthesis earlier and therefore, are closer to the S phase than resting lymphocytes. It is concluded that lymphocytes stimulated in the absence of lysine are activated even though no overall increase in macromolecular synthesis is observed. Furthermore, the kinetics of DNA synthesis following reversal of inhibition by phenylalanine suggests that lymphocytes stimulated during phenylalanine deprivation become arrested at most six hours before S. These results indicate that amino acid deficiencies lead to arrest of activated lymphocytes at various stages of stimulation, depending on how stringent these deficiencies are.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

Chromatin-directed Ribonucleic Acid Synthesis: A Comparison of Chromatins Isolated from Healthy, Avirulent Agrobacterium tumefaciens Inoculated, and Crown-Gall Tumor Tissues of Vicia faba

Chromatin was extracted from healthy, avirulent Agrobacterium tumefaciens inoculated, and crown-gall tumor Vicia faba internodes of the same age. Chromatin from crown-gall tissue produced 5 times more RNA per 100 micrograms of DNA than chromatin from the healthy tissue. When template availability was compared using chromatin with saturating amounts of Escherichia coli RNA polymerase, chromatin from crown-gall tissue had 36% more available template than the controls. In addition, when γ-³²P-ATP was incorporated into the RNA synthesizing reaction mixture, with saturating amounts of E. coli RNA polymerase, there were twice as many RNA chain starts in tumor as in control tissue.     

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Rat liver chromatin which has been briefly sonicated is fractionated by treatment with low concentrations of magnesium ion. At 1.5 mm Mg²⁺, where approximately 20–25% of the chromatin remains soluble after low-speed centrifugation, chemical and physical analysis of the Mg-soluble and Mg-insoluble chromatin fractions show that the fractions possess markedly different properties. The Mg-soluble chromatin has more protein and RNA than the Mg-insoluble chromatin. The histone composition of the two fractions as shown by electrophoretic analysis is similar, but many of the acidic proteins are qualitatively and quantitatively different. The molecular weight of the Mg-soluble chromatin is less than that of the insoluble chromatin based on sedimentation behavior and gel filtration experiments. The soluble chromatin has nearly twice the template activity for RNA synthesis in vitro with added RNA polymerase as the Mg-insoluble chromatin and contains approximately 80% of the in vivo rapidly labeled RNA found in the total chromatin preparation. In addition the Mg-soluble chromatin has a significantly greater amount of “accessible” DNA (62%) as measured by polylysine binding than Mg-insoluble chromatin (48%). The data suggest that (a) fractionation of chromatin preparations can be achieved by titration with Mg²⁺, and (b) chromatin soluble in low concentrations of Mg²⁺ may be enriched in actively transcribed portions of the genome.     

Inhibition of Auxin-induced Deoxyribonucleic Acid Synthesis and Chromatin Activity by 5-Fluorodeoxyuridine in Soybean Hypocotyl

Rootless soybean (Glycine max) seedlings were used as a test system to examine the action of auxin on chromatin-directed RNA synthesis. Chromatin from the basal tissue of rootless seedlings (both control and auxin-treated) had RNA synthetic capacity similar to that of chromatin from comparably treated intact seedlings. When DNA synthesis normally induced in the basal tissue by auxin was blocked in the rootless seedlings by 5-fluorodeoxyuridine, the auxin enhancement of chromatin activity was inhibited 70%. This level was still three times the control level, indicating that auxin influenced the synthetic activity of existing DNA template. Experiments with Escherichia coli RNA polymerase revealed that chromatin from both auxin- and auxin plus 5-fluorodeoxyuridine-treated tissue saturated at higher levels than chromatin from control tissue.     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.