Size and structure of mitochondrial DNA from Physarum polycephalum

One band of DNA with a buoyant density of 1.688 g cm⁻³ is found when isolated mitochondria of the slime mold Physarum polycephalum are incubated with deoxyribonuclease prior to lysis. The DNA consisted mainly of linear molecules up to about 20 μm in length. As many as 10% of the molecules were, however, of open circular conformation with a circumference of 19.1±0.5 μm. Mild lysis conditions favoured the isolation of DNA/protein complexes with no visible free ends. The data suggest that the mitochondrial DNA (mtDNA) of Physarum is a circle and that circularity may be maintained by DNA/protein interaction.     

Zooplankton Growth,Respiration and Grazing on the Australian Margins of the Tropical Indian and Pacific Oceans

The specific activity of aminoacyl-tRNA synthetases (spAARS), an index of growth rate, and of the electron transport system (spETS), an index of respiration, was measured in three size fractions (73–150 μm, >150 μm and >350 μm) of zooplankton during five cruises to tropical coastal waters of the Kimberley coast (North West Australia) and four cruises to waters of the Great Barrier Reef (GBR; North East Australia). The N-specific biomass of plankton was 3–4-fold higher in the Kimberley than on the GBR in all 3 size classes: Kimberley 1.27, 3.63, 1.94 mg m^-3; GBR 0.36, 0.88 and 0.58 mg m^-3 in the 73–150 μm, >150 μm and >350 μm size classes, respectively. Similarly, spAARS activity in the Kimberley was greater than that of the GBR: 88.4, 132.2, and 147.6 nmol PPi hr^-1 mg protein ^-1 in the Kimberley compared with 71.7, 82.0 and 83.8 nmol PPi hr^-1 mg protein ^-1 in the GBR, for the 73–150 μm, >150 μm and >350 μm size classes, respectively. Specific ETS activity showed similar differences in scale between the two coasts: 184.6, 148.8 and 92.2 μL O₂ hr^-1 mg protein^-1 in the Kimberley, against 86.5, 88.3 and 71.3 μL O₂ hr^-1 mg protein^-1 in the GBR. On the basis of these measurements, we calculated that >150 μm zooplankton grazing accounted for 7% of primary production in the Kimberley and 8% in GBR waters. Area-specific respiration by >73 μm zooplankton was 7-fold higher in the Kimberley than on the GBR and production by >150 μm zooplankton was of the order of 278 mg C m^-2 d^-1 in the Kimberley and 42 mg C m^-2 d^-1 on the GBR. We hypothesize that the much stronger physical forcing on the North West shelf is the principal driver of higher rates in the west than in the east of the continent.     

Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.     

Structure of Mitochondrial Dna From Paramecium Tetraurelia*

Mitochondrial DNA (mtDNA) from endosymbiote-free stocks of Paramecium tetraurelia was isolated by 2 procedures. the buoyant density of the mtDNA in neutral CsCI was 1.702 gm/cm³. a value consistent with the melting temperature of the mtDNA. Only linear molecules were observed by electron microscopy. These molecules were homogeneous in size with a monomer molecular weight of 25.6 × 10⁶ daltons. the size of the mtDNA determined after digestion with the restriction endonucleases EcoRI or Hind III agreed with the value obtained by electron microscopy. These studies also revealed that the digestion pattern of mtDNA from stock 172 differed from that of the other 3 stocks (51, 127. 203) examined. Some mtDNA molecules exhibited snapback reassociation following denaturation.     

Penetration of Rhizopus oligosporus into Soybeans in Tempeh

Histological observations were made on the penetration of hyphae of Rhizopus oligosporus into soybean cotyledons in tempeh, an Indonesian soybean food. Hyphal penetrations averaged one per 1,400 μm² (±390 μm²) on the curved (outer) cotyledon surface and one per 1,010 μm² (±340 μm²) on the flat (inner) one. Hyphae infiltrated to a depth of 742 μm, or about 25% of the average width of a soybean cotyledon. This previously unreported degree of penetration offers partial explanation for the rapid physical and chemical changes in soybeans during tempeh fermentation.     

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).     

Chemical and physical properties of adeno-associated satellite virus DNA produced during coinfection with herpes simplex virus

Infectious DNA from adeno-associated satellite virus (ASV) has been isolated from cells coinfected with a temperature-sensitive mutant of herpes simplex virus (HSV) type 1 in the absence of contaminating HSV DNA. This satellite virus DNA does not appear to differ in its physical, chemical and biological properties from DNA isolated directly from virions or from cells co-infected with adenovirus. The DNA is double-stranded with a buoyant density of 1.718 gm/cm³. It sediments at 16S in both neutral and alkaline sucrose gradients. Single-stranded DNA from alkaline sucrose gradients has a modal length of 1.5 μm and demonstrates evidence of internal redundancies in the electron microscope.     

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm²) or H₂O₂ (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1^−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.     

The Anisotropic Elastic Properties of the Sarcolemma of the Frog Semitendinosus Muscle Fiber

Tension and curvature of the sarcolemmal tube of the frog muscle fiber were measured at different extensions and were used to calculate the anisotropic elastic properties of the sarcolemma. A model was derived to obtain the four parameters of the elasticity matrix of the sarcolemma. Sarcolemmal thickness was taken as 0.1 μm. Over the range of reversible sarcolemmal tube extension, the longitudinal elastic modulus E_L = 6.3 × 10⁷ dyn/cm², the circumferential modulus E_c = 0.88 × 10⁷ dyn/cm², the longitudinal Poisson's ratio σ_L = 1.2, and the circumferential Poisson's ratio σ_c = 0.18. At tubular rest length E_L = 1.2 × 10⁷ dyn/cm². The sarcolemma is less extensible in the longitudinal direction along the fiber axis than in the circumferential direction. It can be extended reversibly to 48% of its rest length, equivalent to extending the intact fiber from a sarcomere length of 3 μm to about 4.5 μm. The sarcolemma does not contribute to intact fiber tension at fiber sarcomere lengths <3 μm, and between 3 and 4 μm its contribution is about 20%. It also exerts a pressure on the myoplasm, which can be calculated by means of the model. The longitudinal elastic modulus of the whole fiber is 1 × 10⁵ dyn/cm² at a sarcomere length of 2.33 μm.     

Chloroplast DNA from three archegoniates

1. DNA from female and male Sphaerocarpos donnellii (liverwort) plants exhibits at least two species with buoyant densities of 1.703 (main band) and 1.691 (satellite) g cm^-3 in CsCl equilibrium gradients. At least part, if not all, of the satellite DNA is localized in plastids. It consists of up to 90% of uniformly sized circular molecules of an average circumference of 38.5 m. Compared to other Chlorophyta, the liverwort's cpDNA is unusually low both in diensity and contour length. — 2. On the hand, cpDNA from the ferns Asplenium nidus and Pteris vittata resembles those of higher plants in buoyant density (1.697 g cm^-3) and circumference (about 44.8 m). — 3. Analysis of DNA from the archegoniate chloroplasts with restriction endonucleases indicates chat the cyclic molecules are monomers. — 4. The results show that the circular molecules found in cpDNA of higher plants do not represent the functionally required minimum size of DNA in plastids.Abbreviations cpDNA chloroplast - DNA nucDNA=nuclear - DNA Sal I=restriction endonuclease from Streptomyces albus S - Eco RI restriction endonuclease from Escherichia coli, carrying resistance factor 1 - DTT dithiothreitol (Cleland's reagent) - Saline-EDTA 0.15 M NaCl, 0.1 M ethylene diamine tetraacetic acid, pH 8.0 - SSC 0.15 M NaCl, 0.015 M Na citrate, pH 7.2 - DNAase deoxyribonuclease - Md Megadalton Dedicated to the memory of Prof. Dr. Edgar Knapp     

Assessment of Microbial Fouling in an Ocean Thermal Energy Conversion Experiment

A project to investigate biofouling, under conditions relevant to ocean thermal energy conversion heat exchangers, was conducted during July through September 1977 at a site about 13 km north of St. Croix (U.S. Virgin Islands). Seawater was drawn from a depth of 20 m, within the surface mixed layer, through aluminum pipes (2.6 m long, 2.5-cm internal diameter) at flow velocities of about 0.9 and 1.8 m/s. The temperature of the seawater entering the mock heat exchanger units was between 27.8 and 28.6°C. After about 10 weeks of exposure to seawater, when their thermal conductivity was reported to be significantly impaired, the pipes were assayed for the accumulation of biological material on their inner surfaces. The extent of biofouling was very low and independent of flow velocity. Bacterial populations, determined from plate counts, were about 10⁷ cells per cm². The ranges of mean areal densities for other biological components were: organic carbon, 18 to 27 μg/cm²; organic nitrogen, 1.5 to 3.0 μg/cm²; adenosine 5′-triphosphate, 4 to 28 ng/cm²; carbohydrate (as glucose in the phenol assay), 3.8 to 7.0 μg/cm²; chlorophyll a, 0.2 to 0.8 ng/cm². It was estimated from the adenosine 5′-triphosphate and nitrogen contents that the layer of live bacteria present after 10 weeks was only of the order of 1μm thick. The C/N ratio of the biological material suggested the presence of extracellular polysaccharidic material. Such compounds, because of their water-retaining capacities, could account for the related increase in thermal resistance associated with the pipes. This possibility merits further investigation, but the current results emphasize the minor degree of biofouling which is likely to be permissible in ocean thermal energy conversion heat exchangers.     

Properties of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichia coli.

Summary The tetracycline resistance (Tc) and raffi nose-hydrogen sulfide (Raf-Hys) characters of Escherichia coli D1021 are located on two compatible, conjugative fi^– plasmids named pRSDI and pRSD2, respectively. These were transferred together or separately to the isogenic background of E. coli K12 and isolated as transconjugants. Plasmid molecules isolated as covalently closed circular DNA have been analysed by buoyant density centrifugation, sucrose gradient sedimentation and electron microscopy. pRSDI (Tc) showed a buoyant density of 1.716 g/cm³ (56% GC), the open circular (OC) form had a sedimentation coefficient of 37s. If grown without tetracycline prior to isolation the contour length of most pRSDI molecules was 14.6 (±0.1) m (30.3 × 106 daltons) with a minor species of 13.9 (± 0.1) m owing to a small deletion. Pronounced length variation of pRSDI by growth in the presence of tetracycline owing to gene amplification is subject of a subsequent paper. In contrast, pRSD2 (Raf-Hys) molecules are stable under all conditions tested. The buoyant density was 1.713 g/cm³ (53% GC), the sedimentation coefficient of OGDNA was 58s and the contour length was 40.2 ± 0.6 m (83 × 106 daltons). During exponential growth one copy of pRSD2 per chromosome was found indicating stringent control of plasmid replication. At the onset of stationary growth the copy number rose from one to four per host chromosome indicating relaxation of the replication control. The level of plasmid-coded -galactosidase increases with copy numbers and has been used to test the gene dosage.     

Separation of Marine Bacteria according to Buoyant Density by Use of the Density-Dependent Cell Sorting Method

The purpose of this study was to test whether some phylogenetic groups of natural marine bacteria have unique buoyant densities that allow them to be separated by the density-dependent cell sorting (DDCS) method. We first concentrated a natural bacterial assemblage to collect sufficient numbers of cells. They were separated into three fractions by DDCS, and the community structure in each was clarified by fluorescence in situ hybridization. The cells of Archaea tended to appear in the high-density fraction, whereas those of Cytophaga-Flavobacterium-Bacteroides were in the low-density fraction. We also calculated the sedimentation velocities of three typical marine bacteria (low density, middle density, and high density) using their buoyant density. The sedimentation velocities were approximately 10, 20, and 30 μm h⁻¹; these velocities have ecological implications when the heterogeneity of bacteria is considered at a microscale. To our knowledge, this is the first report on the buoyant density of natural marine bacteria.     

Size of Suspended Bacterial Cells and Association of Heterotrophic Activity with Size Fractions of Particles in Estuarine and Coastal Waters

The size of bacteria and the size distribution of heterotrophic activity were examined in estuarine, neritic, and coastal waters. The data indicated the small size of suspended marine bacteria and the predominance of free-living cells in numerical abundance and in the incorporation of dissolved amino acids. The average per-cell volume of suspended marine bacteria in all environments was less than 0.1 μm³. Cell volume ranged from 0.072 to 0.096 μm³ at salinities of 0 to 34.3‰ in the Newport River estuary, N.C., and from 0.078 to 0.096 μm³ in diverse areas of the Gulf of Mexico. Thus, the free-living bacteria were too small to be susceptible to predation by copepods. In the Newport River estuary, ca. 93 to 99% of the total number of cells and 75 to 97% of incorporated tritium (from ³H-labeled mixed amino acids) retained by a 0.2-μm-pore-size filter passed through a 3.0-μm-pore-size filter. Although the amino acid turnover rate per cell was higher for the bacteria in the >3.0-μm size fraction than in the <3.0-μm size fraction, the small number of bacteria associated with the >3.0-μm size particles resulted in the low relative contribution of attached bacteria to total heterotrophic activity in the estuary. For coastal and neritic samples, collected off the coast of Georgia and northeast Florida and in the plume of the Mississippi River, 56 to 98% of incorporated label passed through a 3.0-μm-pore-size filter. The greatest activity in the >3.0-μm fraction in the Georgia Bight was at nearshore stations and in the bottom samples. Our data were consistent with the hypothesis that resuspension of bottom material is an important factor in influencing the proportion of heterotrophic activity attributable to particle-associated bacteria.     

Differences in SOM Decomposition and Temperature Sensitivity among Soil Aggregate Size Classes in a Temperate Grasslands

The principle of enzyme kinetics suggests that the temperature sensitivity (Q₁₀) of soil organic matter (SOM) decomposition is inversely related to organic carbon (C) quality, i.e., the C quality-temperature (CQT) hypothesis. We tested this hypothesis by performing laboratory incubation experiments with bulk soil, macroaggregates (MA, 250–2000 μm), microaggregates (MI, 53–250 μm), and mineral fractions (MF, <53 μm) collected from an Inner Mongolian temperate grassland. The results showed that temperature and aggregate size significantly affected on SOM decomposition, with notable interactive effects (P<0.0001). For 2 weeks, the decomposition rates of bulk soil and soil aggregates increased with increasing incubation temperature in the following order: MA>MF>bulk soil >MI(P <0.05). The Q₁₀ values were highest for MA, followed (in decreasing order) by bulk soil, MF, and MI. Similarly, the activation energies (E_a) for MA, bulk soil, MF, and MI were 48.47, 33.26, 27.01, and 23.18 KJ mol⁻¹, respectively. The observed significant negative correlations between Q₁₀ and C quality index in bulk soil and soil aggregates (P<0.05) suggested that the CQT hypothesis is applicable to soil aggregates. Cumulative C emission differed significantly among aggregate size classes (P <0.0001), with the largest values occurring in MA (1101 μg g⁻¹), followed by MF (976 μg g⁻¹) and MI (879 μg g⁻¹). These findings suggest that feedback from SOM decomposition in response to changing temperature is closely associated withsoil aggregation and highlights the complex responses of ecosystem C budgets to future warming scenarios.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.     

Recombination in yeast mitochondrial DNA

When haploid yeast strains containing mitochondrial DNAs (mtDNAs) of different buoyant densities are mated, the resulting zygotes contain a mixed population of mitochondria and mitochondrial DNAs. During vegetative growth of diploid cells formed from such a cross between a petite strain with mtDNA of density 1.677 g cm^?3 and a respiratory competent strain with mtDNA of density 1.684 g cm^?3, mtDNAs with intermediate buoyant densities are obtained. Virtually all newly synthesized mtDNA in diploid ρ^? progeny has the intermediate buoyant density. Therefore, within 2 generations of growth of the diploid cells, the intermediate buoyant density species predominate. In crosses between a respiratory competent strain and other petite strains with different values of genetic suppressiveness, it was found that the amount of recombination yielding mtDNAs of intermediate buoyant densities roughly parallels the degree of suppressiveness. Individual clones of respiratory deficient cells from such crosses were also isolated to confirm that stable mtDNAs with intermediate buoyant densities were obtained. Thus, it is apparent that some form of recombination takes place within the mtDNAs of yeast cells that results in stable mtDNA species.     

Extracellular Vesicles of the Hyperthermophilic Archaeon “Thermococcus onnurineus” NA1T

Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, “Thermococcus onnurineus” NA1^T, were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 g cm⁻³, were observed after CsCl ultracentrifugation. The two major EV bands, B (buoyant density at 25°C [ρ²⁵] = 1.2434 g cm⁻³) and C (ρ²⁵ = 1.2648 g cm⁻³), were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120- and 190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. Mapping of the T. onnurineus NA1^T EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1^T were packaged into EVs, except for an ∼9.4-kb region from TON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 3′-terminal sequence and the absence of the 5′-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1^T genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.