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.     

Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40–75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [³H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.     

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.     

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.     

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.     

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.     

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.     

Further investigations on the property of chromatins from apical and axial parts of cauliflower inflorescence

To evaluate the importance of histone F1 for RNA synthesis in cauliflower in relation to the differentiation between apical and axial tissues, the F1 fraction from apical chromatin was removed and the property of the residual (F1-depleted) chromatin was investigated compared to that of axial chromatin. It was found that the derivative melting profile of the residual chromatin was quite similar to that of the axial chromatin, and the nucleotide composition of RNA synthesized with the residual chromatin as template was also like that of the axial chromatin. The phosphate content of the histone F1 fraction isolated from apical chromatin was markedly lower than that of the F1 fraction from axial chromatin. Further, it was ascertained from MAK-column chromatography of [¹⁴C]uridine incorporation into RNA synthesized in excised tissues that axial chromatin in situ has a much higher template activity for RNA synthesis than does apical chromatin. It was postulated that the higher RNA synthesizing activity in the axial part of the cauliflower head can be attributed mainly to the presence of active chromatin in which the histone F1-depleted region is more prevalent than it is in apical chromatin.     

Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

Effects of retinoic acid on protein synthesis in cultured melanoma cells

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G₁ phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis anlaysis of L-[³⁵S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Studies on the role of RNA synthesis in auxin induction of cell enlargement

Selective inhibitors were used to study the connection between nucleic acid synthesis and indoleacetic acid (IAA) induction of cell enlargement. Actinomycin D (act D) and azaguanine (azaG) almost completely inhibit IAA-induced growth in aged artichoke tuber disks when they are added simultaneously with IAA. In contrast, when they are added 24 hours after the hormone, these inhibitors have little or no effect on the induced growth which continues for 48 hours or more with little or no inhibition. Inhibitors of protein synthesis still stop growth when applied 24 hours after the IAA, thus protein synthesis and presumably supporting metabolism are still essential.

In corn coleoptile sections auxin-induced growth did not show any pronounced tendency to become less sensitive to act D as the IAA treatment progressed. Act D did not completely inhibit the response to IAA unless the sections were pretreated with act D for 6 hours. In contrast to act D, cordycepin produced almost complete inhibition of IAA-induced growth when added with the IAA.

Although IAA has a very large and very rapid stimulatory effect (within 10 min) on incorporation of ³²P-orthophosphate into RNA in disks, it did not cause a detectable change in the base composition of the RNA synthesized. Furthermore, the promotive effect could be accounted for through increased uptake of the ³²P. That much of the RNA synthesis in these tissues is not necessary for auxin action is indicated by the results with fluorouracil (FU). FU strongly inhibits RNA synthesis, probably acting preferentially on ribosomal RNA synthesis, without inhibiting auxin-induced growth in the disks or coleoptile sections. FU also strongly inhibited respiration in auxin-treated disks indicating that the large promotion of respiration by auxin likewise may not be entirely necessary for growth.

At least in the artichoke disks, RNA synthesis is required for auxin induction of cell enlargement and not for cell enlargement itself.

The possible relationships of auxin induction of cell enlargement and RNA synthesis are discussed.

     

PROTEIN and RNA SYNTHESES and PRECURSOR UPTAKE WITH ISOLATED NERVE and GLIAL CELLS

Protein and RNA syntheses were investigated with bulk isolated nerve and glial cells from rabbit brain. For polypeptide synthesis, ‘intact’ cells were incubated with [³H]leucine under various conditions and the results were compared with those of polyribosomal polypeptide synthesis. For RNA synthesis ‘intact’ cells were incubated with [³H]uridine or [³H]guanosine and the results were compared with those of DNA-dependent RNA polymerase assay. The bulk isolated ‘intact’ nerve cells were more active in protein synthesis than the ‘intact’ glial cells, while the latter synthesized RNA more actively than the former, although both polyribosomal polypeptide synthesis and DNA-dependent RNA polymerase activity were higher with the nerve cells, indicating a higher potential for the nerve cells. The observed discrepancy of RNA synthesis was explained by the significantly less active uptake of nucleosides with the nerve cells. Both protein and RNA syntheses with ‘intact’ cells were sensitive to hypoxic or glucose-deficient conditions. While both the nerve and glial cells were sensitive to hypoxia to a similar extent, the nerve cells were more sensitive to glucose deficiency. It was suggested that the bulk isolated nerve and glial cells still retain certain integral cell functions as viable cells, and can be utilized for various physiological and pharmacological investigations provided caution is exercised in their application and in the interpretation of the results.