Synthesis of (+) and (−) RNA molecules of potato spindle tuber viroid (PSTV) in isolated nuclei and its impairment by transcription inhibitors

1985 ◽  
Vol 5 (3) ◽  
pp. 251-265 ◽  
Author(s):  
Ellen Spiesmacher ◽  
Hans-Peter Mühlbach ◽  
Martin Tabler ◽  
Heinz L. Sänger
Keyword(s):  
Actinomycin D ◽  
Nuclear Dna ◽  
Potato Spindle Tuber Viroid ◽  
Rna Polymerases ◽  
Cell Nuclei ◽  
Isolated Nuclei ◽  
Rna Molecules ◽  
Entire Process ◽  
Transcription Inhibitors ◽  
Potato Cell

Transcription studies with highly purified potato cell nuclei in combination with a ‘transcription-hybridization analysis’ unequivocally demonstrate that the nucleus is the subcellular site where the entire process of PSTV replication takes place. Inhibition experiments with actinomycin D and α-amanitin furthermore suggest that the nuclear DNA-dependent RNA polymerases I and II are involved in the synthesis of PSTV (+) and (−) RNA, respectively.

scholarly journals DNA Measurement of Overlapping Cell Nuclei in Thick Tissue Sections

1997 ◽  
Vol 14 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Liang Ji ◽  
James Tucker
Keyword(s):  
Liver Tissue ◽  
Nuclear Dna ◽  
Selection Procedure ◽  
Normal Liver ◽  
Cell Nuclei ◽  
3D Segmentation ◽  
Isolated Nuclei ◽  
Tissue Sections ◽  
The Individual ◽  
Dna Measurements

The paper describes an improved image analysis procedure for measuring the DNA content of cell nuclei in thick sections of liver tissue by absorption densitometry. Whereas previous methods only permitted the analysis of isolated nuclei, the new technique enables both isolated and overlapping nuclei to be measured. A 3D segmentation procedure determines whether each object is an isolated nucleus or a pair of overlapping nuclei; in the latter case the combined optical density is redistributed to the individual nuclei. A selection procedure ensures that only complete nuclei are measured.The method has been tested on specially‐prepared Feulgen‐stained 20μsections of normal liver tissue. The overall distribution of the nuclear DNA measurements shows well‐defined diploid and tetraploid peaks, with coefficient of variations of less than 10%. Similar distributions were obtained from both the isolated nuclei and overlapped nuclei sub‐populations.

scholarly journals Involvement of deoxyribonucleic acid polymerase β in nuclear deoxyribonucleic acid synthesis

1978 ◽  
Vol 173 (1) ◽  
pp. 309-314 ◽  
Author(s):  
T R Butt ◽  
W M Wood ◽  
E L McKay ◽  
R L P Adams
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Dna Polymerase Beta ◽  
Cell Nuclei ◽  
High Molecular Weight Dna ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

scholarly journals STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE

1974 ◽  
Vol 63 (2) ◽  
pp. 665-674 ◽  
Author(s):  
V. Mareš ◽  
B. Schultze ◽  
W. Maurer
Keyword(s):  
Young Adult ◽  
Purkinje Cell ◽  
Nuclear Dna ◽  
Brain Regions ◽  
Nuclear Volume ◽  
High Dose ◽  
Prenatal Period ◽  
Cell Nuclei ◽  
Grain Number ◽  
Adult Mice

Neurons of the mouse were labeled with [3H]thymidine during their prenatal period of proliferation. The 3H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the 3H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [3H]ß self-absorption in the material of the sections. Injection of a high dose of [3H]thymidine into young adult mice did not result in 3H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments.

scholarly journals Actinomycin D Induces Histone γ-H2AX Foci and Complex Formation of γ-H2AX with Ku70 and Nuclear DNA Helicase II

2005 ◽  
Vol 280 (10) ◽  
pp. 9586-9594 ◽  
Author(s):  
Hannah Elisabeth Mischo ◽  
Peter Hemmerich ◽  
Frank Grosse ◽  
Suisheng Zhang
Keyword(s):  
Complex Formation ◽  
Actinomycin D ◽  
Nuclear Dna ◽  
Dna Helicase ◽  
Dna Helicase Ii

RNA Polymerases and Controlling Factors from Plant Cell Nuclei

1974 ◽  
pp. 279-293 ◽  
Author(s):  
B. B. Biswas ◽  
H. Mondal ◽  
A. Ganguly ◽  
Asis Das ◽  
R. K. Mandal
Keyword(s):  
Plant Cell ◽  
Controlling Factors ◽  
Rna Polymerases ◽  
Cell Nuclei

The nuclear DNA-dependent ribonucleic acid polymerases of the dermatophytic fungus Microsporum gypseum

1976 ◽  
Vol 22 (2) ◽  
pp. 177-181
Author(s):  
Barbara C. Dill ◽  
J. J. Stock
Keyword(s):  
Ribonucleic Acid ◽  
Nuclear Dna ◽  
Rna Polymerases ◽  
Microsporum Gypseum ◽  
Nuclear Extracts ◽  
Eukaryotic Organisms ◽  
Enzyme Species

The DNA-dependent RNA polymerases of the dermatophytic fungus Microsporum gypseum were partially characterized. Nuclear extracts prepared from vegetative mycelia were fractionated by DEAE-Sephadex chromatography into three enzyme species which resembled in most of their characteristics those of other eukaryotic organisms.

scholarly journals Role of nuclear glycogen synthase and cytoplasmic UDP glucose pyrophosphorylase in the biosynthesis of nuclear glycogen in HD33 Ehrlich-Lettré ascites tumor cells.

1981 ◽  
Vol 89 (3) ◽  
pp. 475-484 ◽  
Author(s):  
C Granzow ◽  
M Kopun ◽  
H P Zimmermann
Keyword(s):  
Tumor Cells ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Membrane System ◽  
Ascites Tumor ◽  
Cell Nuclei ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Isolated Nuclei ◽  
Glycogen Synthase Activity

Biochemical and autoradiographic evidence show both glycogen synthesis and the presence of glycogen synthase (UDP glucose [UDPG]: glycogen 4-alpha-D-glucosyltransferase; EC 2.4.1.11) in isolated nuclei of Ehrlich-Lettré mouse ascites tumor cells of the mutant subline HD33. 5 d after tumor transplantation, glycogen (average 5-7 pg/cell) is stored mainly in the cell nuclei. The activity of glycogen synthase in isolated nuclei is 14.5 mU/mg protein. At least half of the total cellular glycogen synthase activity is present in the nuclei. The nuclear glycogen synthase activity exists almost exclusively in its b form. The Km value for (a + b) glycogen synthase is 1 x 10(-3) M UDPG, the activation constant is 5 x 10(-3) M glucose-6-phosphate (Glc-6-P). Light and electron microscopic autoradiographs of isolated nuclei incubated with UDP-[1-3H]glucose show the highest activity of glycogen synthesis not only in the periphery of glycogen deposits but also in interchromatin regions unrelated to detectable glycogen particles. Together with earlier findings on nuclear glycogen synthesis in intact HD33 ascites tumor cells (Zimmermann, H.-P., V. Granzow, and C. Granzow. 1976. J. Ultrastruct. Res. 54:115-123), the results of tests on isolated nuclei suggest a predominantly appositional mode of nuclear glycogen deposition, without participation of the nuclear membrane system. In intact cells, synthesis of UDPG for nuclear glycogen synthesis depends on the activity of the exclusively cytoplasmic UDPG pyrophosphorylase (UTP: alpha-D-glucose-1-phosphate uridylyltransferase; EC 2.7.7.9). However, we conclude that glycogen synthesis is not exclusively a cytoplasmic function and that the mammalian cell nucleus is capable of synthesizing glycogen.

scholarly journals The loss of rapidly labelled ribonucleic acid from isolated HeLa cell nuclei

1969 ◽  
Vol 112 (1) ◽  
pp. 71-79 ◽  
Author(s):  
J. W. Watts
Keyword(s):  
Hela Cell ◽  
Incubation Medium ◽  
Sedimentation Coefficient ◽  
Cell Nuclei ◽  
Isolated Nuclei ◽  
Bivalent Cations ◽  
Low Concentrations ◽  
Nuclear Rna ◽  
And Diffusion

1. The loss of nucleic acids and protein from isolated HeLa-cell nuclei was studied. During 4hr. incubation at 37° DNA was conserved, but appreciable amounts of RNA and protein were lost. 2. Two classes of nuclear RNA were distinguished: at least 75% of the RNA was lost from the nuclei relatively slowly through degradation to acid-soluble fragments; the rest of the RNA was lost much more rapidly, not only through degradation to acid-soluble fragments but also through diffusion of RNA out of the nuclei into the incubation medium. 3. The RNA that was preferentially lost was the fraction of nuclear RNA that was rapidly labelled when intact HeLa cells were grown in a medium containing radioactive precursors of RNA. 4. The RNA appearing in the incubation medium was apparently partially degraded and had a sedimentation coefficient of about that of transfer RNA. 5. Both the degradation of RNA and the loss of RNA from the nuclei were sensitive to bivalent cations. Low concentrations of Mg2+ and Mn2+ greatly increased the rate of degradation of the rapidly labelled RNA to acid-soluble fragments, and produced a corresponding decrease in the amount of RNA diffusing into the medium. At higher concentrations they suppressed both degradation and diffusion of RNA. The cations Ca2+, Cu2+, Zn2+ and Ni2+ all progressively inhibited both forms of loss of RNA. 6. Salts of univalent cations produced appreciable effects only at ionic strengths of about 0·2, when degradation to acid-soluble fragments was preferentially inhibited. 7. Both ADP and ATP inhibited loss of RNA at about 30mm. 8. It was concluded that the diffusion of rapidly labelled RNA out of the isolated nuclei was not related to the movement of RNA from nucleus to cytoplasm in vivo, but reflected the ease with which the rapidly labelled RNA detached from the chromatin and the permeability of the membranes of isolated nuclei.

DNA–Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis

Genome ◽  
2004 ◽  
Vol 47 (3) ◽  
pp. 559-564 ◽  
Author(s):  
Ellen M Rasch ◽  
Carol Eunmi Lee ◽  
Grace A Wyngaard
Keyword(s):  
Somatic Cell ◽  
Genome Size ◽  
Dna Content ◽  
Nuclear Dna ◽  
Growth Characteristic ◽  
Nuclear Dna Content ◽  
Cell Nuclei ◽  
Chromatin Diminution ◽  
Eurytemora Affinis ◽  
Genomic Studies

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen–DNA cytophotometry to determine a diploid or 2C genome size of 0.6–0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.Key words: copepod, genome size, DNA–Feulgen, calanoid, Eurytemora.

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