scholarly journals LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

1967 ◽  
Vol 33 (3) ◽  
pp. 489-496 ◽  
Author(s):  
J. C. H. de Man ◽  
N. J. A. Noorduyn
Keyword(s):  
Orotic Acid ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Electron Microscopic ◽  
Granular Component ◽  
Hepatic Cells ◽  
Progressive Loss ◽  
Nucleolar Rna ◽  
Silver Grains ◽  
Electron Microscopic Radioautography

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.

scholarly journals REPOPULATION OF POSTMITOTIC NUCLEOLI BY PREFORMED RNA

1973 ◽  
Vol 58 (1) ◽  
pp. 54-63 ◽  
Author(s):  
David M. Phillips ◽  
Stephanie Gordon Phillips
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Normal Morphology ◽  
L929 Cells ◽  
Full Cell ◽  
Nucleolar Rna ◽  
Mitotic Cells

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.

The Effect of Actinomycin D in Vivo Upon Peripheral Nucleoli and Other Nuclear Organelles in Oocytes of Triturus Cristatus

1972 ◽  
Vol 10 (3) ◽  
pp. 833-855
Author(s):  
M. H. L. SNOW
Keyword(s):  
Phase Contrast ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Triturus Cristatus ◽  
Granular Component ◽  
Azure B ◽  
Final Maturation ◽  
Ribosomal Precursor ◽  
20 Nm

Exposure of the ovaries of Triturus cristatus to actinomycin D at a concentration of 100 µg/ml causes characteristic changes in the peripheral nucleoli and other nuclear organelles in oocytes of 0.6-1.1 mm diameter. Viewed with the light microscope untreated oocytes contain nucleoli that stain uniformly with a variety of dyes. They also appear homogeneous tmder phase-contrast optics. After 2 or 4 h of in vivo incubation with actinomycin D, oocyte sections stained with Haidenhain's haematoxylin or viewed under phase-contrast optics show nucleoli composed of 2 regions. The more heavily stained or contrasted zone is crescent-shaped and directed away from the nuclear membrane. Neither sections stained with azure B bromide nor gallocyanin chrome alum show this feature. Ribonuclease digestion does not eliminate or alter it. Autoradiography with [3H]uridine indicates that all recently synthesized RNA is lost from the nucleolus during actinornycin D treatment. The zonation is not therefore a reflexion of RNA distribution. During recovery from actinomycin D poisoning there is a reduction in the degree of zonation shown by nucleoli which re-establish a normal appearance some 48 h after treatment. Electron microscopy of peripheral nucleoli in oocytes sampled during this treatment indicates that the zonation is not associated with reorganization of ultrastructural components. During incubation with actinomycin D the coarse granules (20 nm diameter) are completely lost from the nucleolus. There is associated shrinkage of the nucleolus which after treatment is found to consist entirely of fibrils (5 nm thick) and small granules. The reappearance of the coarse granules during recovery is completed in about 48 h. It is thought that the loss of the granular component during treatment represents the movement of the 30-S precursor and the 18-s ribosomal unit from the nucleolus. Some 20-30 µm inside the nucleus of untreated oocytes is a region containing many spheroidal bodies, less than 1.0 µm diameter. They have been termed micronucleoli and consist of granules 2.5-5 nm in diameter and fibrils of similar thickness. Actinomycin D treatment causes these components to segregate and eventually (within 24 h of treatment) the granular component is extruded. This component reappears during the second day after treatment. It is postulated that these micronucleoli represent the sites at which the 30-S ribosomal precursor undergoes its final maturation. The segregation of components induced by actinomycin D is probably the morphological manifestation of an abnormal metamorphosis of this precursor. Treatment with actinomycin D also induces the immediate formation within the nucleus of crystalline bodies composed of lamellae 16 nm wide, 4 nm thick and with a centre-to-centre spacing of 8-10 nm. They are not present 24 h after treatment. They are thought to represent a protein fraction normally associated with periods of intense RNA synthesis.

scholarly journals THE DISORGANIZATION OF HEPATIC CELL NUCLEOLI INDUCED BY ETHIONINE AND ITS REVERSAL BY ADENINE

1968 ◽  
Vol 36 (2) ◽  
pp. 313-328 ◽  
Author(s):  
Hisashi Shinozuka ◽  
Peter J. Goldblatt ◽  
Emmanuel Farber
Keyword(s):  
Electron Microscope ◽  
Nuclear Membrane ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Hepatic Cell ◽  
Short Term ◽  
Hepatic Cells ◽  
Basic Reaction ◽  
Reaction Patterns

The structure of nuclei and nucleoli of hepatic cells after short-term ethionine administration was investigated with the electron microscope. By 1½ hr after the injection, a distinct alteration occurred in the nucleoli which was characterized by the appearance of electron-opaque masses in the nucleolonema. After 6–8 hr, the nucleoli showed partial fragmentation into small, dense masses. Large aggregates of interchromatinic granules appeared in the nucleoplasm. Condensation of chromatin became prominent in the nucleoplasm particularly along the nuclear membrane. By 12 hr almost complete fragmentation of nucleoli had occurred. The administration of adenine or methionine at 4 hr prevented the development of nucleolar changes. Also, adenine administration at 8 hr after ethionine completely reversed the nucleolar lesion by 12 hr. After methionine administration at 8 hr, many nucleoli showed incomplete reconstruction with many twisted ropelike structures when viewed 4 hr later. Identical structures were found when adenine was given at 8 hr, and animals were sacrificed 2 hr later. On the basis of this observation, the simplified structures of nucleoli found 2 hr after adenine or 4 hr after methionine appeared to be precursors of the nucleolonema. It is suggested that nucleoli show at least two basic reaction patterns to inhibitors of RNA synthesis, one typified by actinomycin D and one by ethionine.

scholarly journals NUCLEOLAR AND PERICHROMOSOMAL RNA SYNTHESIS DURING MEIOTIC PROPHASE IN THE MOUSE TESTIS

1974 ◽  
Vol 60 (1) ◽  
pp. 39-53 ◽  
Author(s):  
A. L. Kierszenbaum ◽  
Laura L. Tres
Keyword(s):  
Sex Chromosomes ◽  
Meiotic Prophase ◽  
Rna Synthesis ◽  
Transcriptional Activity ◽  
Mouse Testis ◽  
Synthetic Activity ◽  
Synthetic Cell ◽  
Nucleolar Organizers ◽  
Nucleolar Rna ◽  
Silver Grains

The transcriptional activity during meiotic prophase in the mouse testis is studied with light microscopy and high-resolution autoradiographic techniques using [3H]uridine as a labeled precursor. In the present study, two types of RNA synthesis are detected during meiotic prophase: an extranucleolar RNA synthesis of perichromosomal localization and a nucleolar RNA synthetic activity. In some of the autosomes and close to the basal knobs, the activity of the nucleolar organizers is evidenced by the incorporation of [3H]uridine into nucleolar masses from zygotene on and at earlier labeling times. The evolution of nucleoli and the formation of a nucleolus attached to the sex pair are described during the different meiotic stages. Perichromosomal labeling, from leptotene on, reaches a maximum during middle pachytene and falls progressively to a low level at longer incorporation times. Sertoli's cell, the most active RNA synthetic cell in the seminiferous epithelium, rises to a maximum of labeling and drops at earlier times compared with the meiotic prophase cells. The condensed sex chromosomes show some scattered silver grains especially at middle pachytene. The axial chromosome cores and synaptonemal complexes are devoid of silver grains during the meiotic prophase. The observations suggest that a control mechanism operates during meiotic prophase to regulate transcriptional activity in the sex chromosomes and to provide differential RNA synthesis in autosomal bivalents at various stages of prophase and within certain segments of the chromosomes.

Sites of RNA synthesis and migration in the adrenal cortex cells of the young rat, as shown by radioautography

1983 ◽  
Vol 102 (4) ◽  
pp. 594-602 ◽  
Author(s):  
Maria C. Magalhães ◽  
M. M. Magalhães ◽  
A. Coimbra
Keyword(s):  
Adrenal Cortex ◽  
Rna Synthesis ◽  
Zona Fasciculata ◽  
Time Intervals ◽  
Granular Component ◽  
Sequence Of Events ◽  
Young Rat ◽  
And Migration ◽  
Fibrillar Component ◽  
Silver Grains

Abstract. The sites of RNA synthesis, processing and migration were studied with light and electron microscope radioautography in adrenal zona fasciculata cells of the young rat. Animals were injected iv with [3H]uridine and sacrificed at various time intervals. A marked radioactive peak was found in blood at the 20 min interval, later time intervals being considered as chase periods. Labelling was high over nucleoli of fasciculata cells at 1 h, with the silver grains concentrated over the fibrillar component. Simultaneous though lower peaks occurred over the condensed chromatin and the interchromatin space. Nucleolar and extranucleolar values were decreased at 8 and 24 h, most silver grains overlaying the cytoplasm at this time period. At 8 h, the granular component of the nucleolus was more heavily labelled than the fibrillar component. This sequence of events suggests a relatively slow uptake of the labelled nucleotides and slow formation of pre-rRNA molecules in the fibrillar component of the nucleolus, with the processing into smaller subunits occurring in the granular component during initial chase. Extranucleolar RNAs were synthesized simultaneously but in a smaller rate. Most nucleolar RNA was finally transferred to the cytoplasm. This pattern will serve as a basis for studying the effects of ACTH on the different steps of RNA synthesis and transport in adrenal cortex cells.

Bovine abnormal preimplantation embryos: analysis of segregated cells occurring in the subzonal space and/or blastocoele cavity for their nuclear morphology and persistence of RNA synthesis

Zygote ◽  
2002 ◽  
Vol 10 (2) ◽  
pp. 141-147 ◽  
Author(s):  
J. Pivko ◽  
P. Grafanau ◽  
E. Kubovičová
Keyword(s):  
Rna Synthesis ◽  
Actinomycin D ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Transcription Inhibition ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Labelling Intensity ◽  
Intense Labelling

Bovine embryos in the early blastocyst/blastocyst stage were analysed by [5-3H]uridine labelling followed by electron microscopic autoradiography. In normal control embryos an intact zona pellucida, evenly developed blastomeres and a transparent perivitelline space were seen. In this group, the blastomeres of the trophoblast and embryoblast showed high homogeneous labelling localised in the nucleoplasm and even more intense labelling in the nucleolus. On the contrary, in addition to evident cytoplasmic disintegration, a clearly different labelling pattern and a low labelling intensity were observed in the nuclei of the segregated cells in the subzonal space and in those free in the blastocoele cavity. A typical nuclear morphological feature of these blastomeres was chromatin marginalisation, similar to that observed in embryos treated with actinomycin D for transcription inhibition. It is concluded that the segregated cells are arrested in their further differentiation.

scholarly journals LA SYNTHÈSE DE L'ADN MITOCHONDRIAL CHEZ TETRAHYMENA PYRIFORMIS

1968 ◽  
Vol 39 (2) ◽  
pp. 369-381 ◽  
Author(s):  
Renée Charret ◽  
Jean André
Keyword(s):  
Mitochondrial Dna ◽  
Great Majority ◽  
Tetrahymena Pyriformis ◽  
Logarithmic Phase ◽  
Time Interval ◽  
Electron Microscopic ◽  
Mitochondrial Population ◽  
A Cell ◽  
Silver Grains ◽  
Electron Microscopic Radioautography

Electron microscopic radioautography has been used to study the synthesis of mitochondrial DNA after incorporation of thymidine-3H by cultures in logarithmic phase of Tetrahymena pyriformis during periods ranging from 15 min to 12 hr. The great majority of silver grains are distributed over the macronuclei, the micronuclei, and the mitochondria. The intensity of the label over the entire mitochondrial population is a function of the length of the incubation period within the time interval considered. The intensity of the mitochondrial label was compared with that of the nuclear label. Mitochondria incorporate at the same rate whether the nuclei are synthesizing or not. This persistence of mitochondrial incorporation in the absence of nuclear incorporation excludes the hypothesis of a nuclear origin for mitochondrial DNA. We are not able to determine whether the apparent continuity of synthesis in the entire mitochondrial population of a cell actually represents a series of asynchronous discontinuities.

scholarly journals Appendix: Radioautographic localization of labelled proteins after incubation of guinea-pig islets of Langerhans with [3H]tryptophan

1974 ◽  
Vol 140 (1) ◽  
pp. 22-23 ◽  
Author(s):  
S. L. Howell ◽  
C. Hellerström ◽  
M. Whitfield
Keyword(s):  
B Cells ◽  
Guinea Pig ◽  
B Cell ◽  
Islets Of Langerhans ◽  
Electron Microscopic ◽  
Storage Granules ◽  
Silver Grains ◽  
Electron Microscopic Radioautography ◽  
Significant Concentration

An analysis was made by electron-microscopic radioautography of the distribution of silver grains over storage granules of A2 and B cells after incubation of isolated guinea-pig islets of Langerhans for 17h in the presence of [3H]tryptophan. A significant concentration of labelled proteins was present in the A2 cell, but not in the B-cell granules.

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