Mitochondrial DNA from 4-Cell Stages of Ascaris Lumbricoides

1974 ◽  
Vol 16 (3) ◽  
pp. 593-601
Author(s):  
H. TOBLER ◽  
C. GUT
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Dna ◽  
Ascaris Lumbricoides ◽  
Buoyant Density ◽  
Contour Length ◽  
Cell Stage ◽  
Replicative Intermediates ◽  
Dna Amounts

Mitochondrial DNA (mtDNA) has been isolated from 4-cell stages of Ascaris lumbricoides. This DNA amounts to about 40% of the total quantity of 4-cell-stage DNA. Its buoyant density in neutral CsCl gradients is 1.686 g cm-3. Electron microscopy of mtDNA demonstrated the presence of circular molecules with an average contour length of 4.64 µm. About 15% of these molecules are supercoiled, covalently closed circles, whereas some 2% consist of double-forked circular molecules. The form and size of these branched molecules suggest that they are replicative intermediates.

scholarly journals ACCUMULATION OF REPLICATIVE INTERMEDIATES OF MITOCHONDRIAL DNA IN TETRAHYMENA PYRIFORMIS GROWN IN ETHIDIUM BROMIDE

1974 ◽  
Vol 61 (2) ◽  
pp. 383-397 ◽  
Author(s):  
William B. Upholt ◽  
Piet Borst
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Tetrahymena Pyriformis ◽  
Linear Molecule ◽  
Chain Growth ◽  
Predominant Species ◽  
Velocity Gradients ◽  
Large Numbers ◽  
Replicative Intermediates

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10–15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.

scholarly journals SINGLE STRAND-CONTAINING REPLICATING MOLECULES OF CIRCULAR MITOCHONDRIAL DNA

1973 ◽  
Vol 56 (1) ◽  
pp. 230-245 ◽  
Author(s):  
David R. Wolstenholme ◽  
Katsuro Koike ◽  
Patricia Cochran-Fouts
Keyword(s):  
Mitochondrial Dna ◽  
Single Strand ◽  
Molecular Forms ◽  
Contour Length ◽  
Chick Embryos ◽  
Single Stranded Region ◽  
Mitochondrial Dnas ◽  
Replicative Intermediates ◽  
Circular Contour ◽  
Protein Monolayer

Mitochondrial DNAs (mtDNAs) from Chang rat solid hepatomas and Novikoff rat ascites hepatomas were examined in the electron microscope after preparation by the aqueous and by the formamide protein monolayer techniques. MtDNAs from both tumors were found to include double-forked circular molecules with a form and size suggesting they were replicative intermediates. These molecules were of two classes. In molecules of one class, all three segments were apparently totally double stranded. Molecules of the second class were distinguished by the fact that one of the segments spanning the region between the forks in which replication had occurred (the daughter segments) was either totally single stranded, or contained a single-stranded region associated with one of the forks. Daughter segments of both totally double-stranded and single strand-containing replicating molecules varied in length from about 3 to about 80% of the circular contour length of the molecule. Similar classes of replicating molecules were found in mtDNA from regenerating rat liver and chick embryos, indicating them to be normal intermediates in the replication of mtDNA All of the mtDNAs examined included partially single-stranded simple (nonforked) circular molecules. A possible scheme for the replication of mtDNA is presented, based on the different molecular forms observed

scholarly journals EVIDENCE FOR DISCONTINUOUS REPLICATION OF CIRCULAR MITOCHONDRIAL DNA MOLECULES FROM NOVIKOFF RAT ASCITES HEPATOMA CELLS

1974 ◽  
Vol 61 (1) ◽  
pp. 14-25 ◽  
Author(s):  
Katsuro Koike ◽  
David R. Wolstenholme
Keyword(s):  
Mitochondrial Dna ◽  
Hepatoma Cells ◽  
Single Strand ◽  
Contour Length ◽  
Rat Tissues ◽  
Ascites Hepatoma ◽  
Mean Values ◽  
Mitochondrial Fractions ◽  
Rat Ascites Hepatoma ◽  
Replicative Intermediates

Double-forked circular molecules of mitochondrial DNA (mtDNA) from rat tissues, indicated by their form and size to be replicative intermediates, are of two structurally distinct classes. Molecules of the first class are totally double stranded. Molecules of the second class are defined by one daughter segment being totally or partially single stranded. Length histograms of daughter segments measuring between 2% and 44% of the total 5-µm molecular contour were constructed from samples of both classes of replicating molecules derived from mtDNA or Novikoff rat ascites hepatoma cells. For single strand-containing molecules, the lengths fell into eight distinct, reproducible groups with mean values separated by 4.1–7.6% of the circular contour length. For totally double stranded molecules, the lengths fell into seven groups, corresponding to seven of the groups found for single strand-containing molecules. These results suggest that along at least 44% of the contour of mtDNA molecules there exist discrete points at which DNA synthesis tends to be arrested. This may indicate that there are pauses in normal mtDNA synthesis. However, as the DNA used in these experiments was isolated from mitochondrial fractions, the findings may indicate that continuation of synthesis beyond specific points on the nucleotide strands requires a factor which is not available after cell disruption.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Studies of Circular DNA Using a New Electron Microscopic Technique

1973 ◽  
Vol 31 ◽  
pp. 596-597
Author(s):  
C. N. Gordon
Keyword(s):  
Electron Microscopy ◽  
Aqueous Salt Solution ◽  
Contour Length ◽  
Cleavage Surface ◽  
Mica Substrate ◽  
Electron Microscopic ◽  
Replica Technique ◽  
Circular Dna ◽  
Transmission Electron ◽  
Electron Microscopic Technique

Gordon and Kleinschmidt have described a new preparative technique for visualizing DNA by electron microscopy. This procedure, which is a modification of Hall's “mica substrate technique”, consists of the following steps: (a) K+ ions on the cleavage surface of native mica are exchanged for Al3+ ions by ion exchange. (b) The mica, with Al3+ in the exchange sites on the surface, is placed in a dilute aqueous salt solution of DNA for several minutes; during this period DNA becomes adsorbed on the surface. (c) The mica with adsorbed DNA is removed from the DNA solution, rinsed, dried and visualized for transmission electron microscopy by Hall's platinum pre-shadow replica technique.In previous studies of circular DNA by this technique, most of the molecules seen were either broken to linears or extensively tangled; in general, it was not possible to obtain suitably large samples of open extended molecules for contour length measurements.

Existence of DNA in yeast microbodies

1978 ◽  
Vol 36 (2) ◽  
pp. 232-233
Author(s):  
Masako Osumi ◽  
Misuzu Nagano ◽  
Hiroko Kazama
Keyword(s):  
Catalase Activity ◽  
Buoyant Density ◽  
Model System ◽  
Contour Length ◽  
Native State ◽  
Normal Alkane ◽  
Remarkable Increase ◽  
Dna Molecule ◽  
Mitochondrial Dnas

We have found that microbodies appeared profusely together with a remarkable increase in catalase activity in normal alkane-grown cells of hydrocarbon-utilizing Candida yeasts, and that the microbodies multiplied by division in these cells. These features of Candida yeasts seem to provide a useful model system for studies on the biogenesis of the microbody. Subsequently, we have succeeded in isolation of Candida microbodies in an apparently native state, as judged biochemically and morphologically. The presence of DNA in the purified microbody fraction thus obtained was proved by the diphenylamine method. DNA molecule of about 15 urn in contour length was released from an isolated microbody. The physicochemical analyses of the microbody DNA revealed that its buoyant density differed from nuclear and mitochondrial DNAs. All these results lead us to the possibility that there is a novel type of DNA in microbodies.

Nucleic Acid Molecules Prepared by Monolayer Techniques

1972 ◽  
Vol 30 ◽  
pp. 178-179
Author(s):  
Dimitrij Lang
Keyword(s):  
Electron Microscopy ◽  
Standard Deviation ◽  
Nucleic Acid ◽  
Cytochrome C ◽  
Nucleic Acids ◽  
Contour Length ◽  
Sample Standard Deviation ◽  
Molecular Weights ◽  
Length Measurements ◽  
Protein Monolayer

The success of the protein monolayer technique for electron microscopy of individual DNA molecules is based on the prevention of aggregation and orientation of the molecules during drying on specimen grids. DNA adsorbs first to a surface-denatured, insoluble cytochrome c monolayer which is then transferred to grids, without major distortion, by touching. Fig. 1 shows three basic procedures which, modified or not, permit the study of various important properties of nucleic acids, either in concert with other methods or exclusively:1) Molecular weights relative to DNA standards as well as number distributions of molecular weights can be obtained from contour length measurements with a sample standard deviation between 1 and 4%.

Ultrastructural and cytological observations of apothecial tissues of Geopyxis carbonaria (Pezizales, Ascomycetes)

1990 ◽  
Vol 68 (2) ◽  
pp. 243-257 ◽  
Author(s):  
James W. Kimbrough ◽  
Jack L. Gibson
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Key Words ◽  
Mother Cell ◽  
Secondary Wall ◽  
Cell Stage ◽  
Wall Structures ◽  
Transmission Electron ◽  
Secondary Wall Formation ◽  
Characteristic Features

Cytological observations are made on apothecial tissues of Geopyxis carbonaria, using transmission electron microscopy. Characteristic features of both the medullary and ectal excipula are examined. Changes in ascus apex and wall structures are examined during ascus ontogeny, especially in relation to operculum position and structure. Ultrastructure of septum configuration is observed and compared in the excipulum, ascogenous hyphae, paraphyses, and at the base of young asci. Ascosporogenesis is observed from the ascus mother cell stage and initial spore delimitation until secondary wall formation. The cytological and ultrastructural observations on this species are discussed in relation to their possible taxonomic or phylogenetic value. Key words: ascosporogenesis, Discomycetes, ascospore ultrastructure, septal ultrastructure, cytochemistry.

Fate of genetically marked mitochondrial DNA from spermatocytes microinjected into mouse zygotes

Zygote ◽  
1999 ◽  
Vol 7 (2) ◽  
pp. 151-156 ◽  
Author(s):  
James M. Cummins ◽  
Hidefumi Kishikawa ◽  
Denise Mehmet ◽  
Ryuzo Yanagimachi
Keyword(s):  
Mitochondrial Dna ◽  
Germ Cells ◽  
Restriction Site ◽  
Mutual Recognition ◽  
Nuclear Genome ◽  
Pcr Amplification ◽  
Cell Stage ◽  
Mt Dna ◽  
Mouse Zygotes ◽  
Host Embryo

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 × DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4–5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.

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