Evidence For a Polarized Movement of the Lateral Loops of Newt Lampbrush Chromosomes During Oogenesis

1969 ◽  
Vol 5 (1) ◽  
pp. 1-25
Author(s):  
M. H. L. SNOW ◽  
H. G. CALLAN

Actinomycin D inhibits RNA synthesis on the lateral loops of newt lampbrush chromsomes. Partial inhibition does not provoke marked morphological alteration of ordinary lateral loops, most of which recover to the full their capacity for RNA synthesis within 2 days of treatment. However, occasional ordinary loops do not recover completely within the first few days after treatment, and in such loops RNA-synthesizing capacity is restricted to a region adjoining the thinner insertion in the parent chromomere. A greater degree of inhibition of RNA synthesis is accompanied by loss of matrix from ordinary lateral loops, and in the extreme case the loop axes retract to their parent chromomeres and neighbouring chromomeres coalesce; for the ordinary loops, full recovery from this stripped condition is nevertheless possible. Some 20 µ per loop extends during the first day following exposure to actinomycin, and normal morphology and RNA-synthesizing capacity are regained within 2-4 days. The giant granular loop of Triturus cristatus cristatus chromosome XII responds to extreme actinomycin D poisoning in different fashion. Matrix does not at once slough off its loop axis, but the loop present at the time of treatment is progressively replaced by a new granular loop which develops between the parent chromomere and the original loop's dense tip. These observations support the theory that the DNA-containing axes of all lateral loops of lampbrush chromosomes continually extend from their parent chromomeres, engage in RNA synthesis while extended, and carry the associated RNP matrix along as they move towards the return insertions in the parent chromomeres, where loop axis retraction occurs.

1973 ◽  
Vol 58 (1) ◽  
pp. 54-63 ◽  
Author(s):  
David M. Phillips ◽  
Stephanie Gordon Phillips

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.


1972 ◽  
Vol 10 (3) ◽  
pp. 833-855
Author(s):  
M. H. L. SNOW

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.


Author(s):  
Awtar Krishan ◽  
Dora Hsu

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).


1973 ◽  
Vol 134 (4) ◽  
pp. 1103-1113 ◽  
Author(s):  
A. Betteridge ◽  
M. Wallis

The effect of insulin on the incorporation of radioactive leucine into growth hormone was investigated by using rat anterior pituitary glands incubated in vitro. A 50% stimulation over control values was observed at insulin concentrations above 2μm (280munits/ml). The effect was specific for growth hormone biosynthesis, over the range 1–5μm-insulin (140–700munits/ml). Lower more physiological concentrations had no significant effect in this system. Above 10μm (1.4 units/ml) total protein synthesis was also increased. The stimulation of growth hormone synthesis could be partially blocked by the addition of actinomycin D, suggesting that RNA synthesis was involved. Insulin was found to stimulate the rate of glucose utilization in a similar way to growth hormone synthesis. 2-Deoxyglucose and phloridzin, which both prevented insulin from stimulating glucose utilization, also prevented the effect of insulin on growth hormone synthesis. If glucose was replaced by fructose in the medium, the effect of insulin on growth hormone synthesis was decreased. We conclude that the rate of utilization of glucose may be an important step in mediating the effect of insulin on growth hormone synthesis.


1974 ◽  
Vol 20 (7) ◽  
pp. 977-980 ◽  
Author(s):  
David K. Horowitz ◽  
Peter J. Russell

Sexual differentiation in male strains of the aquatic fungus Achlya ambisexualis Raper is induced by antheridiol, a sexual steroid hormone secreted by female strains. Antheridiol-induced initiation of the morphologically distinct antheridial branches in male Achlya is completely prevented when DNA-dependent RNA synthesis is inhibited by actinomycin D. In addition antheridial branch elongation is inhibited to a degree proportional to the concentration of actinomycin D added. Thus, evidence indicates that RNA synthesis is required for antheridiol-induced initiation of antheridial branching and that continued RNA synthesis is required for elongation of antheridial branches.


1993 ◽  
Vol 120 (2) ◽  
pp. 277-290 ◽  
Author(s):  
J Sommerville ◽  
J Baird ◽  
B M Turner

Lampbrush chromosomes from oocytes of the amphibian Triturus cristatus have been used to examine the role of histone acetylation in transcription by indirect immunofluorescence with antisera to H4 acetylated at specific lysine residues. Electrophoresis on acid-urea-Triton gels and Western blotting have confirmed the specificity of these antisera and defined the order in which particular lysine residues are acetylated in amphibian cells. As in mammals, lysine 16 is acetylated first, followed by 8 and/or 12 and then 5. With lampbrush chromosomes from immature (previtellogenic) oocytes, antisera to H4 acetylated at lysines 8, 12, and 16 labeled fluorescent foci at the bases of transcription loops. Antisera to H4 acetylated at lysine 5 labeled weakly (i.e., the tri- and tetraacetylated isoforms must be rare). Loops showed weak labeling of the chromatin axis but intense fluorescence at particular points, which probably represent incompletely decondensed chromatin. The RNP matrix of loops, including the RNP-rich sphere bodies and the dense matrix of "marker" loops, was not labeled. Treatment of immature oocytes with butyrate for 12 h to inhibit histone deacetylation did not affect immunolabeling, suggesting that turnover of H4 acetates is slow. In contrast, in chromosomes from mature oocytes, in which loops have retracted and transcription is low, butyrate caused an increase in labeling with all antisera, followed by the appearance of vestigial loops, weakly labeled, but with regions of intense fluorescence. These loops contain RNP and are presumably transcriptionally active. We conclude that H4 acetates turn over more rapidly in mature than immature oocytes and that histone hyperacetylation precedes, and possibly induces, loop formation and transcriptional activation.


1980 ◽  
Vol 41 (1) ◽  
pp. 115-123
Author(s):  
H.G. Callan ◽  
R.W. Old

Denatured 3H-labelled DNAs containing Xenopus or human globin sequences hybridize to RNA transcripts on a single pair of lateral loops on lampbrush chromosome IX of Triturus cristatus carnifex, and to no other loops on this chromosome or the rest of the complement. However they do so, not because of the globin sequences in the probes, but rather because the plasmids from which the probes were prepared were constructed with G.C homopolymer tails. Simple sequence poly d(C/G)n probes also hybridize with RNA transcripts on this same pair of loops, and with no others.


1998 ◽  
Vol 45 (1) ◽  
pp. 127-132 ◽  
Author(s):  
M Piestrzeniewicz ◽  
K Studzian ◽  
D Wilmańska ◽  
G Płucienniczak ◽  
M Gniazdowski

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.


1972 ◽  
Vol 75 (1) ◽  
pp. 11-14 ◽  
Author(s):  
L. Fiume ◽  
I. Nardi ◽  
S. Bucci ◽  
G. Mancino

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.


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