Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein,DNA and cytoplasmic protein in rat liver

Summary From rats fed ad libitum and kept under a 12+12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined every four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphtol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max=0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear nonhistone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

Nuclear non-histone protein content of G0/G1 cells as related to growth fraction in mouse mammary tumors.

In eight mouse mammary tumors with varying growth fractions DNA and non-histone nuclear protein (NHNP) were determined by absorption cytophotometry of Feulgen-Naphthol Yellow S stained, isolated cells. It was found that: 1. The mean NHNP content of cells with postmitotic DNA content (G0 + G1) increased with increasing growth fraction. 2. The mean NHNP content of S and G2 cells in the eight tumors did not vary significantly with growth fraction. 3. The frequency distributions of NHNP in G0/G1 cells were unimodal and right-skewed. The results are interpreted as follows: A) G0 cells differ from G1 cells by their lower content of NHNP. B). If it is assumed that the G0 and G1 compartments are arranged in series, the cells in the transition from G0 to late G1 may account for the unimodality and skewedness of the NHNP frequency distributions of postmitotic cells.     

Quantification of nuclear non-histone proteins by Feulgen-Naphthol Yellow S cytophotometry

Summary Owing to the accumulation of nuclear non-histone protein (NHP) (a) in cells entering the cell cycle from the quiescent state and (b) in continuously cycling cells during G₁ phase, a simultaneous determination of DNA and nuclear NHP is of high potential utility in cell kinetic studies. This paper provides guidelines for a Feulgen-Naphthol Yellow S staining technique for this purpose. It discusses details of the preparation and quantification procedures, and reviews the evidence for a quantitative relationship between nuclear Naphthol Yellow S binding and nuclear NHP.     

Nuclear proteins and DNA in the myocardial myocytes in compensatory cardiac hypertrophy]

Using cytophotometry, the amount of DNA, total nuclear proteins and of histones were studied in the myocardial cells during days 21--36 of experimental compensatory hypertrophy of the heart (in rats). The enlargement of myocardial nuclei during the cardial hyperfunction was accompanied by accumulation of total nuclear protein, in particular, the histone fraction, without distinct changes in DNA. Analysis of correlations between nuclear proteins and DNA in the myocardial cells allows to reveal a delayed accumulation of histones in the big and gigantic nuclei, with a superfluous increase in non-histone nuclear proteins. In middle-sized nuclei, non-histone proteins have little changes against intensive accumulation of histones.     

Feulgen-Naphthol Yellow S cytophotometry of liver cells. The effect of formaldehyde induced shrinkage on nuclear Naphthol Yellow S binding.

1. In isolated liver cells, fixed in 4 per cent formaldehyde (NFS) for Feulgen-Naphthol Yellow S (F-NYS) staining of DNA and protein, nuclear shrinkage increases the nuclear concentration of solids to 46 per cent (w/v) before the start of the NYS staining. 2. When a fixative mixture of methanol:acetic acid:formalin (85:5:10 by volume; MAF) is used, the concentration of nuclear solids during NYS staining remain at a physiological level of 19 per cent. 3. By exposing liver cells to NFS for 10 to 120 seconds before fixation in MAF, increasing nuclear shrinkage can be induced with increasing pretreatment in NFS. Nuclear NYS binding decreases in parallel with the decreasing nuclear volume in cells thus treated. As the shrinkage induced reduction in NYS binding may vary with the net charge of nuclear non-histone proteins, MAF fixation must be preferred for quantitative determinations of nuclear non-histone protein in F-NYS stained, isolated cells. 4. Fixation in MAF offers the same advantages as NFS fixation as regards the small loss of proteins during the Feulgen staining procedure and the excellent reproducibility of the F-NYS staining. Storage of MAF fixed cells in the fixative for a few days does not alter their F-NYS staining properties. 5. In MAF fixed, F-NYS stained cells there is no NYS binding to histone basic amino acid residues.     

Dry mass, DNA and non-histone protein determinations in lung cancer cells

Summary Cell nuclei from two biopsies of bronchial mucosa, seven squamous-cell carcinomas and six small-cell carcinomas of the lung were investigated. DNA and non-histone protein (NHP) content were simultaneously determined in Feulgen—Naphthol Yellow S-stained smears by means of multiple plug cytophotometry. In addition, the nuclear dry mass of cells originating from the same populations was measured by interference microscopy.DNA histograms of carcinomas were characterized by DNA stemlines being situated in the diploid range in four out of six small-cell carcinomas and in the hyperdiploid to hypertetraploid range in six out of seven squamous-cell carcinomas.Polyploid values (up to 8 c) were seldom found in small-cell carcinomas whereas squamous-cell carcinomas showed a broader dispersion approaching the 16 c level.The similarity of the NHP distributions with the DNA histograms was more marked in small-cell carcinomas than in the squamous-cell carcinomas. In comparison with the NHP distributions of normal bronchial epithelial cells, those of carcinomas were shifted to higher values. The increased NHP content was found to be a more prominent sign of malignancy in small-cell carcinomas than the DNA mass. The increase in nuclear dry mass in carcinomas was mainly caused by the accumulation of NHP in tumour cell nuclei.     

Enhanced myocardial RNA synthesis in spontaneously hypertensive rats possible role of high-mobility group non-histone proteins

Cardiac hypertrophy in spontaneously hypertensive rats is associated with increased nuclear RNA polymerase activity. In order to explore mechanisms facilitating the interaction of the enzyme with its endogenous template, we compared the structure of nuclear chromatin from myocytes of 20-week-old spontaneously hypertensive rats and normotensive Wistar-Kyoto controls. Enhanced RNA synthesis in hypertensive rats was accompanied by increased susceptibility to digestion by deoxyribonuclease I. Nick translation of nuclei also resulted in higher nucleotide incorporation in hypertensive rats. Salt-extraction abolished the differences in deoxyribonuclease I sensitivity between the two animal groups. Reconstitution with either 0.35 M NaCl-extract or high mobility group (HMG) non-histone proteins restored digestion susceptibility but did not equalize SHR and WKY cells. SDS-polyacrylamide gel electrophoresis of 0.35 M NaCl-extracts and supernatants from deoxyribonuclease I digestion revealed the presence of HMG proteins which were preferentially released in hypertensive rats. There was a small but statistically significant increase in nuclear HMG protein content in hypertensive rats (0.12 +/- 0.02 mg/mg DNA vs. 0.09 +/- 0.02 mg/mg DNA in Wistar-Kyotos, P less than 0.05) but no difference in their electrophoretic appearance. These results indicate that chromatin structure is altered in the hypertrophied myocardium with resultant increase in deoxyribonuclease I susceptibility. This increase appears to be partly dependent on the high-mobility group non-histone proteins.     

Mitochondrial DNA, RNA and protein synthesis in normal and hypothyroid developing rat liver

Mitochondrial DNA, RNA and protein synthesis in normal and hypothyroid rat liver between the ages of -3 and 21 days were followed. In normal rats DNA polymerase activity and protein synthesis behaved similarly, showing two peaks of activity, one at -3 and the other at 21 days of age. RNA polymerase activity did not change between days -3 and 14, whereas it increased by 21 days of age. Hypothyroidism delayed the developmental pattern of DNA polymerase activity, affected RNA polymerase activity only at 21 days, whereas it inhibited protein synthesis at birth and in the third week of life. The cytochrome aa3 content appeared to be affected by hypothyroidism at birth and at 21 days of age.     

Changes in nuclear and nucleolar protein content during the growth and differentiation of root parenchyma cells in plant species with different DNA-endoreplication dynamics

Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.     

Quantitative cytophotometric determination of DNA, RNA and lysine bound protein in relationship to zygote formation and protein synthesis in myxamoebae and swarm cells of Didymium iridis

Quantitative cytochemical determinations were made of the DNA of zygotes formed from myxamoebae and swarm cells of Didymium iridis. The nuclear and cytoplasmic RNA and lysine bound protein of these cells were also measured. Significant zygote formation in myxamoebae crosses began at 20-30 min, while swarmers required 35-40 min. Myxamoebae, however, demonstrated a greater ability to form zygotes. The total cytoplasmic RNA and protein bound lysine for myxamoebae was higher than that of the swarmer cells. This observed decrease in swarmers may be due to reduced protein synthesis. Values for nuclear RNA were higher in the myxamoebae, but nuclear lysine bound protein was higher in the swarmers. The data presented suggest that prefusion swarmers, after replicating their DNA, go into a period of G2 arrest and remain in this condition postfusion. In contrast, prefusion myxamoebae readily divide after DNA replication, and continue to synthesize nuclear DNA, and to divide after fusion.     

Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in rat liver after protein restriction.

Rats were fed for 6 days on a diet containing either 3 or 20% high-quality protein. Nuclei were isolated from liver and DNA-dependent RNA polymerases (EC 2.7.7.6) extracted with 1 M-(NH4)2SO4. The proteins were then precipitated with 3.5 M-(NH4)2SO4 and after dialysis applied to a DEAE-Sephadex column. The column was developed with a gradient of (NH4)2SO4. Polymerase I separated well from alpha-amanitin-sensitive polymerase II. The enzyme activities were compared between the two dietary groups. Rats that had received 3% protein showed a lower polymerase I activity per g wet wt. of liver, per mg of DNA and per mg of protein. Polymerase II was lower in activity per g wet wt. of liver and per mg of DNA, but was higher per mg of protein. Polyacrylamide-gel electrophoretograms showed a higher proportion of contaminating proteins in polymerase II fractions isolated from 20%-protein-fed rats. The data explain the lower activity obtained per mg of protein in these rats. It is concluded that a decrease in dietary protein content from 20 to 3% induces a fall in content and specific activity of RNA polymerase I and II in liver.     

Alterations of protein synthesis in arbovirus-infected L cells

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

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.