Ram sperm mitochondrial DNA

Circular DNA was isolated from mitochondrial fractions of ram spermatozoa by SDS treatment followed by convex sucrose gradient centrifugation. The DNA had a contour length of 5.0 micron. Its buoyant density was 1.6983 g cm-3, which was smaller than two nuclear DNA components with buoyant densities of 1.6999 and 1.7156 g cm-3, found in ram spermatozoa. The Tm of the mitochondrial DNA was 69.7 degrees C. The mole fraction G+C calculated from the buoyant density and melting temperature was 39.1% and 38.6%, respectively.     

Mitochondrial genetics

Summary Cytoplasmic petite mutants of Saccharomyces cerevisiae carrying the gene conferring the resistance to chloramphenicol on one hand and the gene conferring the resistance to erythromycin on the other, have been crossed with each other. The two types of petites differed in the buoyant densities of their mitochondrial DNA. A novel type of evidence has been adduced, that the two genes are indeed located on mitochondrial DNA. Diploid petite recombinants were found, carrying both genes and containing not a mixture of the two parental DNAs but a new species of mitochondrial DNA of intermediate buoyant density. Recombination of mitochondrial genes involves therefore breakage and reunion of DNA molecules. New suppressiveness, different from the two parental ones, can result from the recombination of mitochondrial DNA. Recombination between petite mutants implies that the mitochondrial recombination enzymes have to be synthesized on cytosol ribosomes.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.     

Selective incorporation of thymidine in the organelle DNA of Polytoma obtusum

Polytoma obtusum was found to selectively incorporate exogenous thymidine into its leukoplast DNA. The nuclear DNA was unable to incorporate [3H]thymidine, although both DNA species could be labeled with radioactive adenine with similar efficiencies. The mitochondrial DNA (mitDNA), which had a buoyant density of 1.714 g/ml and was banded slightly on the heavier side of the nuclear DNA peak, was also found to incorporate a small amount of [3H]thymidine. These observations suggest that P. obtusum lack cytoplasmic thymidine kinase, whereas the enzyme is present in both leukoplast and mitochondria.     

Chromatographic resolution of nucleic acids extracted from Penicillium chrysogenum

Summary High molecular weight DNA extracted from Penicillium chrysogenum has been fractionated using RPC-5 Analog, into three distinct types designated 1, 2 and 3. Types 1 and 2 have the same buoyant density of 1.710 g/cm³ and together appear to comprise the nuclear DNA. Type 1 is enriched for repeated sequences which are normally observed in restriction digests of P. chrysogenum total DNA. Conversely, type 2 appears to be composed entirely of non-repetitive sequences. Type 3 has been identified as mitochondrial DNA, having a buoyant density of 1.695 g/cm³ and an estimated molecular weight of 31.6×10⁶ Daltons.     

Characterization of mitochondrial DNA from Paramecium aurelia with EcoRI and Hae II restriction endonucleases

The mitochondrial DNA from several species of Paramecium aurelia was characterized by its buoyant density, contour length, and cleavage pattern with the restriction endonucleases EcoRI and Hae II. The density and length were the same for all species while the cleavage pattern was unique to each species. In one species the fragments generated by EcoRI were not in equal molar ratios and the amount of an additional fragment appeared to be dependent on the replication stage of the mitochondrial DNA. The uniqueness of each fragmentation pattern was used to identify the mitochondrial DNA in interspecies hybrids.     

Replication of bromodeoxyuridylate-substituted mitochondrial DNA in yeast.

The DNA of several strains of Saccharomyces cerevisiae was labeled by growing the culture in medium supplemented with thymidylate and bromodeoxyuridylate. It was thus possible to follow the course of mitochondrial DNA replication in density shift experiments by determining the buoyant density distribution of unreplicated and replicated DNAs in analytical CsCl gradients. DNA replication was followed for three generations after transfer of cultures from light medium to heavy medium and heavy medium to light medium. Under both conditions, the density shifts observed for mitochondrial DNA were those expected for semiconservative, nondispersive replication. This was further confirmed by analysis of the buoyant density of alkali-denatured hybrid mitochondrial DNA. With this method, no significant recombination between replicated and unreplicated DNA was detected after three generations of growth.     

I-DNA suppressed by Ethidium Bromide and accumulated with Dactinomycin

THE I-DNA in cytoplasmic particles^1–4 of embryonic chick cells has a 260 nm/280 nm ratio of 1.95 (ref. 5) and a buoyant density of CsCl of 1.700–1.703 g/ml.; mitochondrial DNA has a buoyant density of 1.708 g/ml.^6,7. Other reports of non-mitochondrial cytoplasmic DNA different from nuclear DNA have appeared recently^8–10.     

Mitochondrial deoxyribonucleic acid from Tetrahymena pyriformis and its kinetic complexity

1. Mitochondrial DNA from Tetrahymena pyriformis strain T has a buoyant density (rho) of 1.685 compared with rho1.688 for whole cell DNA. Mitochondrial preparations from T. pyriformis strain W show an enrichment of a light satellite (rho1.686), although this is not obtained free from nuclear DNA (rho1.692). 2. T. pyriformis mitochondrial DNA renatures rapidly and the kinetics of this process indicate a complexity of approx. 3x10(7) daltons. 3. The base-pairing in the renaturation product is of a precise nature, since the ;melting' temperature (80.5 degrees C) is indistinguishable from that of the native DNA (80.5 degrees C). 4. Centrifugation of mitochondrial DNA in an alkaline caesium chloride density gradient gives two bands, implying the separation of the complementary strands.     

Characterization of the genome of the basidiomycete Schizophyllum commune

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.     

Simian virus 40 DNA replication in nuclear monolayers.

Simian virus 40 DNA replication has been studied in nuclear monolayers prepared by treatment of monolayers of BSC-1 monkey kidney cells with Nonidet P-40. These nuclear monolayers incorporated [3H]TTP into two types of viral replicative intermediates that sediment as 25-26S and 22-23S species, respectively, in neutral sucrose gradients. The 22-23S species behaves, in dye buoyant density equilibrium gradients, as a late replicative intermediate. Examination of both species in alkaline sucrose gradients revealed the presence of two types of newly synthesized strands: (i) 4-7S strands and (ii) full-length, or nearly full-length, 10-16S strands. At low TTP concentrations (less than 0.5 muM), the two size classes were found in approximately equal amounts. However, at 10 to 50 muM TTP, the proportion of the longer strands increased, with a corresponding decrease in the relative amount of the 4-7S species. Thus, the joining of small, Okazaki-like fragments to the growing chain appears to require a much higher concentration of TTP than the synthesis of the fragments themselves. Replicating simian virus 40 DNA synthesized in the nuclear monolayers is is associated with "M bands", as previously demonstrated for replicating simian virus 40 DNA in cultured whole cells.     

The possible role of endogenous nucleases in the structural organization of chromatin]

Two fractions of rat liver nuclei with different buoyant density have been obtained. The electrophoretic analysis of the oligonucleosome patterns of DNA out of nuclei of these two fractions revealed different levels of activity in endonucleases. In case of inhibition during the extraction of activity in Ca, Mg-dependent endonucleases, the average size of high polymeric DNA is larger for nuclei with bigger buoyant density (fraction I) than for nuclei with smaller ones (fraction II). This finding is evidence of in situ existence of two pools of liver nuclei with different endogenic nuclease activities. In nuclear chromatin fraction I DNA is torsionally stressed; in fraction II it is relaxed that correlates with larger activity of endonucleases and smaller buoyant density of this fraction. A hypothesis on a possible role of endonucleases in chromatin structure organization has been put forward. According to this hypothesis a modulation of activity in nuclear endonucleases can determine different packaging and activity of chromatin from different pools of cellular nuclei.     

The DNA of Acanthamoeba spp.; A Method for Extraction and Its Characterization

SYNOPSIS. A method is described which permits the recovery of a high yield of undegraded DNA from Acanthamoeba castellanii. The DNA of A. castellanii is comprised of 2 species: the buoyant density of the major component is 1.720 and of the minor component, 1.693. The renaturation characteristics of the minor component suggest that it is of mitochondrial origin. Two components are also present in the DNA of A. palestinensis, A. polyphaga and A. astronyxis. The buoyant densities of major and minor components differ between species.     

Detection of a contaminant cell culture line by restriction endonuclease cleavage patterns of mitochondrial DNA.

A putative HeLa cell culture line was discovered to be contaminated with mouse cells by examination of agarose gel profiles of restriction endonuclease digests of mitochondrial DNA. The contamination was confirmed by karyotypic analysis, and by observation of the mouse satellite band in an analytical buoyant density centrifugation of total cellular DNA. Restriction endonuclease analysis of mitochondrial DNA is suggested as a useful method for monitoring the species of cells in culture.     

Isolation and physicochemical characterization of mitochondrial DNA from cultured cells ofPetunia hybrida

Summary Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm^–3 in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2–30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).     

Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes

Three DNA buoyant density species (nuclear, 1.692 g cm⁻³; mitochondria 1.705 g cm⁻³; and proplastid, 1.713 g cm⁻³) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm⁻³ species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis.     

Further characterization of intracytoplasmic A particle-associated DNA.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.     

Deoxyribonucleic acid base composition of the yeastlike and mycelial phases of Histoplasma capsulatum and Blastomyces dermatitidis

The base composition in moles percent guanine plus cytosine (%GC) of both nuclear and mitochondrial deoxyribonucleic acid (DNA) isolated from the yeastlike and mycelial phases of the dimorphic fungal pathogens Histoplasma capsulatum and Blastomyces dermatitidis was determined by techniques of thermal denaturation and CsCl buoyant density gradient equilibrium centrifugation. The mean observed values for GC content of nuclear DNA from H. capsulatum and B. dermatitidis were 47.3 and 48.2%, respectively. What is speculated to be mitochondrial DNA was found to be 34.0% for H. capsulatum and 34.3% for B. dermatitidis. Thermal denaturation curves for Blastomyces DNA indicated a bimodality in thermal denaturation profiles, thereby suggesting a significant mitochondrial DNA contamination. Mitochondrial DNA appeared to represent a smaller percentage of the total DNA prepared from Histoplasma, and was not observed consistently to affect%GC values as determined by thermal denaturation profiles. On the basis of the now known perfect stage of B. dermatitidis (Ajellomyces dermatitidis) as a member of the family Gymnoascaceae, the close approximation of%GC content of nuclear DNA of this fungal organism with that of H. capsulatum suggests possible phylogenetic relationship. It is suggested that the just reported, but as yet unclassified, perfect stage of H. capsulatum may be found to be phylogenetically a primitive form of the Gymnoascaceae.     

Distinction between Nuclear Satellite DNAs and Chloroplast DNA in Higher Plants

Triton X-100 solubilized chloroplast DNA but not nuclear DNA from a mixture of chloroplasts and nuclei. The buoyant density of chloroplast DNA was different from that of the satellite DNA in all of the species examined (Phaseolus coccineus, Cucumis sativus, Cucumis melo, Antirrhinum majus, Vicia faba, Oenothera fruiticosa youngii). Chloroplast DNA constituted between 4.3% and 0.25% of the total leaf DNA in these species, and was present as 5 to 20 copies in each chloroplast.