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

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.

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.

The Inifiation, Maintenance and Termination DNA Synthesis: A Study of Nuclear DNA Replication Using Amoeba Proteus As a Cell Model

1971 ◽  
Vol 9 (1) ◽  
pp. 1-21
Author(s):  
M. J. ORD
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Thymidine Incorporation ◽  
Cell Model ◽  
Amoeba Proteus ◽  
Early Prophase ◽  
Precursor Pool ◽  
Endogenous Precursor ◽  
The Individual

By means of the nuclear transfer technique for amoeba, combinations of nuclei and cytoplasma from all parts of the cell cycle were available for examining the individual roles of the nucleus and cytoplasm in nuclear DNA replication. Neither S-phase nor division sphere cytoplasm proved capable of initiating a new round of nuclear DNA synthesis in the G2 nucleus. There was some indication that G2 nuclei which were transferred into early prophase cells, i.e. before the formation of a regular division sphere, did incorporate more [3H]thymidine than control G2 nuclei. Positive proof of the induction of DNA synthesis in ‘immature’ nuclei was observed in only two cases. When young G2 nuclei were transplanted into late G2 amoebae, the addition of the donor nucleus generally resulted in the older nucleus being held in a late G2 phase until the younger nucleus passed through its G2. Division of 90% of heterophasic homokaryons was synchronous, with a subsequent synchrony of DNA synthesis. A study of variance in [3H]thymidine incorporation by S nuclei sharing the same cytoplasm - using binucleate, trinucleate and multinucleate homokaryons - showed that nuclei through the peak-S period synthesized DNA at approximately similar rates. The large differences in [3H]thymidine incorporation by nuclei of amoebae of equal age appear due to differences in endogenous precursor pools. These would vary both with differences in food intake and with the draining of remote precursor pools for simultaneous cellular activities, particularly RNA synthesis. When sharing the same cytoplasm nuclei in peak S incorporated similar amounts of [3H]thymidine. Though cytoplasm did not influence the progress of DNA replication by a nucleus, it did influence the use of exogenous [3H]thymidine by the cell, and in so doing caused much of the variation observed in the labelling of nuclei during S. Nuclei sharing the same cytoplasm, and so subject to the same precursor pool changes, incorporated similar amounts of exogenous thymidine. Once DNA synthesis had been initiated it continued to completion regardless of the cytoplasm which surrounded it. Thus neither the maintenance nor termination of DNA synthesis required a special cytoplasmic state.

The localization of (3H) thymidine incorporation in the DNA of replicating spinach chloroplasts by electron-microscope autoradiography

1976 ◽  
Vol 20 (2) ◽  
pp. 341-355
Author(s):  
R.J. Rose ◽  
J.V. Possingham
Keyword(s):  
Grain Size ◽  
Electron Microscope ◽  
Thymidine Incorporation ◽  
Membrane System ◽  
Chloroplast Envelope ◽  
Lamellar System ◽  
Electron Microscope Autoradiography ◽  
Spinach Chloroplasts ◽  
Microscope Autoradiography ◽  
Leaf Disks

Electron-microscope autoradiography has been used to obtain information on the localization of DNA labelled with [3H]thymidine in chloroplasts known to be replicating and concomitantly synthesizing and segregating DNA, in cultured leaf disks. The studies were made using both Microdol-X developer and a ‘compact’ developer which gave a smaller grain size. About 80% of the grains were associated with the granal membranes and with presumptive DNA regions (3-nm fibril material in clear areas). Few grains occurred in association with the chloroplast envelope. We suggest that the DNA of chloroplasts is associated with the grana lamellae and extends into the stroma. Some light-microscope autoradiographs of whole chloroplasts show spiral or helical-like labelling patterns. We interpret these patterns as demonstration of the possibility that DNA occurs along the length of a continuous lamellar membrane system. Chloroplast fractionation experiments provided data consistent with the electron-microscope autoradiographic studies as most of the label was associated with chlorophyll-containing lamellae. We consider an association of chloroplast DNA molecules along the length of a continuous lamellar system would ensure an orderly segregation of DNA to daughter chloroplasts, during the binary fission of spinach chloroplasts by constriction division.

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

A STUDY OF BLASTOCYSTS DURING DELAY AND SUBSEQUENT IMPLANTATION IN LACTATING MICE

1968 ◽  
Vol 42 (3) ◽  
pp. 453-463 ◽  
Author(s):  
ANNE McLAREN
Keyword(s):  
Dna Synthesis ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Uterine Horn ◽  
Uterine Epithelium ◽  
Serial Sections ◽  
Uterine Lumen ◽  
Fresh State

SUMMARY Blastocysts were studied on the 5th and 8th day of pregnancy in lactating mice, in the fresh state, flushed from the uterus, in squash preparations and in serial sections. At the earlier period some mitosis was observed. Tritiated thymidine incorporation studies gave some evidence of DNA synthesis on the 5th and 6th days of pregnancy. By the 8th day the blastocysts were longer, contained more cells, and mitosis had ceased. They were located at the anti-mesometrial end of the uterine lumen, closely apposed to the uterine epithelium, and with their long axes parallel to the long axis of the uterine horn. Implantation could be induced, either by the removal of the litter, or by the injection of an appropriate dose of oestrogen on the 5th or 7th (but not the 4th) day of pregnancy. Both treatments were followed by the appearance of W-bodies in the neighbourhood of the blastocysts, the disappearance of the shed zonae, and the appearance of Pontamine Blue reactivity, oedema of the uterine stroma and formation of the primary decidual zone, in that order.

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