scholarly journals CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS

1964 ◽  
Vol 22 (3) ◽  
pp. 505-513 ◽  
Author(s):  
D. R. Wolstenholme ◽  
W. Plaut
Keyword(s):  
Electron Microscope ◽  
Dna Synthesis ◽  
Light Microscope ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Electron Microscope Autoradiography ◽  
Cytoplasmic Dna ◽  
Microscope Autoradiography ◽  
Normal Particle ◽  
Labeling Pattern

The application of electron microscope autoradiography to Amoeba proteus cells labeled with tritiated thymidine has permitted the identification of morphologically distinct particles in the cytoplasm as the sites of incorporated DNA precursor. The particles correspond to those previously described from light microscope studies, with respect to both H3Tdr incorporation and distribution in centrifugally stratified amoebae. Ingested bacteria differ from the particles, in morphology as well as in the absence of associated label. Attempts to introduce a normal particle labeling pattern by incubating amoebae with labeled sediment derived from used amoeba medium failed. The resultant conclusion, that the particles are maintained in the amoeba by self-duplication, is supported by the presence of particles in configurations suggestive of division.

Timing of nucleolar DNA replication in Amoeba proteus

1976 ◽  
Vol 22 (3) ◽  
pp. 521-530
Author(s):  
I. Minassian ◽  
L.G. Bell
Keyword(s):  
Electron Microscope ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Central Region ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
G2 Phase

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.

scholarly journals CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS

1962 ◽  
Vol 15 (3) ◽  
pp. 525-534 ◽  
Author(s):  
M. Rabinovitch ◽  
W. Plaut
Keyword(s):  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Living Cells ◽  
Large Numbers ◽  
Cytoplasmic Dna ◽  
Basic Dyes ◽  
Nuclease Digestion ◽  
High Degree ◽  
Very High

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detectable structures. Staining experiments with basic dyes, including the fluorochrome acridine orange, revealed the presence of large numbers of 0.3 to 0.5 µ particles in the cytoplasm of all cells studied. The effect of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with H3-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles.

The Cytoplasmic Incorporation of Tritiated Thymidine During Oogenesis in Pteridium Aquilinum

1971 ◽  
Vol 8 (2) ◽  
pp. 467-487
Author(s):  
D. C. SIGEE ◽  
P. R. BELL
Keyword(s):  
Electron Microscope ◽  
Tritiated Thymidine ◽  
Pteridium Aquilinum ◽  
Egg Cell ◽  
Cell Periphery ◽  
Short Term ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Silver Grains ◽  
Stable Molecule

The short-term incorporation of tritiated thymidine into the cytoplasm of cells undergoing oogenesis was investigated in Pteridium aquilinum using electron-microscope autoradiography. There was substantial uptake into the central cell and egg cell during the 6-h labelling period. The quantity and distribution of the label incorporated into the cytoplasm were closely similar in cells fixed immediately after the labelling period and in those immersed for a further 18 h in unlabelled thymidine. This suggested that incorporation was into a stable molecule, with little nucleoside turnover and no subsequent migration within the cytoplasm. Enzyme studies indicated that the tritiated thymidine was incorporated almost entirely into DNA, most probably the DNA undergoing replication. Within the cytoplasm the label was markedly and consistently concentrated in plastids and mitochondria. This localization was not, however, complete and 5-40% was attributable to sites in the ground cytoplasm. A gradient of incorporated label was demonstrated within the cytoplasm in both central cells and egg cells. Concentration was high adjacent to the nucleus and low at the cell periphery. This gradient could be satisfactorily explained by the distribution of the plastids and mitochondria within the cytoplasm, the labelling of the organelles being irrespective of their position. The results of statistical examination of the frequencies of the silver grains associated with the mitochondria and plastids were considered to indicate general uptake of label directly into the DNA of these organelles without nuclear participation.

Cytoplasmic DNA Synthesis at Meiotic Prophase in Lilium henryi

1979 ◽  
Vol 27 (3) ◽  
pp. 273 ◽  
Author(s):  
DR Smyth ◽  
T Shaw
Keyword(s):  
Hydrochloric Acid ◽  
Dna Synthesis ◽  
Meiotic Prophase ◽  
Light Microscope ◽  
Dnase I ◽  
Rnase A ◽  
Chromosomal Dna ◽  
Cytoplasmic Dna ◽  
Microscope Autoradiography ◽  
Small Clusters

Extensive cytoplasmic DNA synthesis has been discovered at meiotic prophase in Lilium henryi. Explanted microsporocytes were cultured in medium containing I3H]thymidine. Light microscope autoradiography revealed many small clusters of grains (< 3 μm) in the cytoplasm of premeiotic and pachytene cells. There were about 10 to 20 clusters per cell in each section. Most of the cyto- plasmic label was extracted by hot hydrochloric acid and DNase I, but not by RNase A or pronase. Thus the grains reflect DNA synthesis, and not incorporation of products of thymidine catabolism which is extensive in this tissue. These localized centres of DNA synthesis, previously unreported, might result from mitochondrial or plastid replication, or from amplification of excised chromosomal DNA.

scholarly journals RESOLUTION IN AUTORADIOGRAPHY USING SEMITHIN SECTIONS

1974 ◽  
Vol 22 (4) ◽  
pp. 217-222 ◽  
Author(s):  
MIRIAM M. SALPETER ◽  
G. C. BUDD ◽  
SHARON MATTIMOE
Keyword(s):  
Electron Microscope ◽  
High Voltage ◽  
Line Source ◽  
Light Microscope ◽  
Electron Microscope Autoradiography ◽  
Density Distributions ◽  
Microscope Autoradiography ◽  
Semithin Sections ◽  
Tritium Labeling

Resolution studies were performed on light microscope autoradiographic specimens of 0.5-1 µ thicknesses coated with Ilford L4 and Kodak NTB2 and AR10 emulsions. A radioactive line source, previously employed for electron microscope autoradiography (Salpeter, Bachmann and Salpeter, 1969; Salpeter and Salpeter, 1971), was used. It was found that with tritium labeling the half-distance (HD) was approximately 3500 Å. This value was not significantly altered with increasing section beyond 0.5 µ or emulsion thickness beyond ~2,000 Å. A 0.5-µ section labeled with 14C had an HD value of 7000 Å when coated with a monolayer of Ilford L4 or Kodak NTB2 emulsion. Increasing section or emulsion thickness did increase this value. It was found that the density distributions, normalized in units of their own HD, gave a good fit to the set of universal curves derived from electron microscope autoradiographic specimens. These universal curves are thus applicable for analyzing light microscope autoradiographic specimens. Some tritium HD values applicable to high voltage microscopy are also given.

Incorporation of Cholesterol into Peripheral Nerve Myelin

1972 ◽  
Vol 30 ◽  
pp. 334-335
Author(s):  
Frank A. Rawlins
Keyword(s):  
Electron Microscope ◽  
Sciatic Nerve ◽  
Electron Microscope Autoradiography ◽  
Myelin Sheaths ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Sciatic Nerves ◽  
Autoradiographic Analysis ◽  
Old Mice ◽  
Short Times

Several speculations exist as to the site of incorporation of preformed molecules into myelin. The possibility that an autoradiographic analysis of cholesterol-1,2-H3 incorporation at very short times after injection might shed some light in the solution of that problem led to the present experiment.Cholesterol-1,2-H3 was injected intraperitoneally into 24 tenday old mice. The animals were then sacrificed at 10,20,30,40,60,90,120 and 180 min after the injection and the sciatic nerves were processed for electron microscope autoradiography. To analyze the grain distribution in the autoradiograms of cross and longitudinal sections from each sciatic nerve myelin sheaths were subdivided into three compartments named: outer 1/3, middle 1/3 and inner 1/3 compartments.It was found that twenty min. after the injection of cholesterol -1.2-H3 (Figs. 1 and 2), 55% of the total number of grains (t.n.g) found in myelin were within the outer 1/3 compartment, 9% were within the middle 1/3 and 36% within the inner 1/3 compartment

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