ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm³ has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 µm; it reassociates with a low C₀t_1/2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm³. MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm³, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.     

Ribonucleic acid and protein content of eukaryotic ribosomes isolated from Artemia salina

We are studying the ribosomes from the cryptobiotic embryos of Artemia salina. Here we report on the relation between the optical density at 260 nm of a ribosome solution and its RNA and protein content. Using the original Lowry method or a modified version, it has been found that 1 A₂₆₀ unit contains 42.4 ± 1.6 μg of protein, and, from the phosphorus content, that the same solution contains 41.6 ± 1.0 μg of RNA. Analytical isodensity equilibrium centrifugation gives a value of 1.570 ± 0.005 g/cm³ for the buoyant density of these ribosomes in CsCl. This density can be related to a protein content of 51%, which is in accord with the chemical determinations. The relation between the optical density of ribosomes, RNA, and protein content and the optical density of rRNA of different systems, such as Escherichia coli, yeast, A. salina, and rat liver is discussed.     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Biophysical studies on double-stranded RNA from turnip yellow mosaic virus-infected plants

Summary Double-stranded RNA was isolated in mg quantities from TYMV-infected cabbage plants by a modified phenol procedure. Chromatography of the RNA on methylated albumin and hydroxyapatite is described. The base composition (A=21.3; U=21.2; G=28.8; C=28.7) was in good agreement with the values expected for a double-stranded RNA consisting of TYMV RNA and a strand complementary to it. The buoyant density of the RNA in Cs₂SO₄ was 1.617 g/cm³. Single-stranded TYMV RNA banded at 1.642 g/cm³ in Cs₂SO₄. The RNA sedimented in the analytical ultracentrifuge with an average sedimentation coefficient of 10–11 s. Absorbance as a function of temperature was determined in several different media. The absorbance-temperature profiles were typical of those expected for double-stranded RNA. Denatured RNA was examined by equilibrium density gradient centrifugation.     

Effect of short schemes on body composition measurements using Air-Displacement Plethysmography

Background

Air-displacement plethysmography (ADP) is becoming a popular method to assess body composition. Several studies have shown certain types of clothing can affect measurements of body density, however no study has specifically investigated the effect of cotton gym shorts and spandex bicycle shorts on body density.

Methods

Thirty-seven males (23.0 ± 3.2 yr., 177.3 ± 5.4 cm., 74.8 ± 7.5 kg.) and thirty-eight females (23.7 ± 5.3 yr., 163.6 ± 8.4 cm., 57.1 ± 7.0 kg.) had their body density measured by ADP in three clothing schemes: 1) a tight fitting Speedo^® swim suit (criterion measure), 2) cotton gym shorts, and 3) spandex bicycle shorts. The clothing was provided by the University of Oklahoma Body Composition Laboratory and the testing schemes were performed in random order.

Results

The regression of body density by the criterion measure against body density while wearing cotton gym shorts for the entire group (y = 0.001 + 0.991x, SEE = 0.003 g/cm³) and the females (y = 0.059 + 0.934x, SEE = 0.003 g/cm³) did not significantly deviate from the line of identity. However in males the regression significantly deviated from the line of identity (y = 0.052 + 0.944x, SEE = 0.002 g/cm³). Body density by the criterion measure and body density while wearing spandex bicycle shorts did not significantly differ from the line of identity for the entire group (y = -0.018 + 1.013x SEE = 0.003 g/cm³), in males (y = -0.002 + 1.001x, SEE = 0.003 g/cm³), or females (y = 0.073 + 0.925x, SEE = 0.003 g/cm³). Residual plot analysis revealed no group or gender bias in either the cotton gym shorts or in the spandex bicycle shorts.

Conclusion

It would appear bicycle spandex shorts are an acceptable alternative to a Speedo^® like swim suit, however we advise that subjects adhere to the strict clothing protocol that is recommended by the manufacturer.

     

Chain growth rate of -galactosidase during exponential growth and amino acid starvation

S_d phage were incubated in 1 m-O-methylhydroxylamine. At various time-intervals, samples of modified phage were isolated and disrupted either by heating or by treatment with detergent. Changes in viscosity and buoyant density of disrupted preparations took place in the course of modification. Three transient synchronous drops in viscosity and buoyant density levels were observed with minima at five minutes, one and three hours of modification. The specific viscosity of the preparations at minima was 10 to 20% that of the disrupted unmodified phage.Properties of the phage preparation isolated during the third period of decreased viscosity were studied in more detail. This preparation, subjected to thermal disruption, gives a single DNA-containing band in Cs₂SO₄ gradient centrifugation corresponding to a buoyant density of 1.37 g/cm³ (cf. 1.39, 1.29 and 1.43 g/cm³ for whole phage, phage ghosts and native phage DNA, respectively).The band contains practically all the ³⁵S label that was present in the starting phage, suggesting that it corresponds to a complex of phage DNA with protein. Electron microscopy revealed complexes as thick strands of 50 to 300 Å diameter bonded to globular particles of varying size.In four hours of modification, the viscosity and buoyant density of disrupted phage returned to values characteristic of unmodified preparations. The DNA band contained no ³⁵S label. Electron microscopy of the substance of this band revealed fibres of 20 Å diameter.A possible explanation of the results is based on the assumption of pre-existing non-covalent interaction of C₍₄₎—NH₂ moieties of cytidine residues with nucleophilic groupings of coating protein within the virion. It is assumed that it is this interaction that holds DNA in “non-native” conformation within intact phage particles and thus explains its peculiar properties discovered earlier. In the present case, the interaction determines the formation of DNA-protein crosslinks under O-methylhydroxylamine treatment via the earlier postulated intermediate product of cytosine modification. Restoration of “normal” physical properties of disrupted phage after more prolonged modification is explained by cleavage of the DNA-protein cross-links due to reaction of the postulated intermediate with O-methylhydroxylamine affording N₍₄₎-methoxy-6-methoxy-amino-5,6-dihydrocytidine residues.     

Characterisation of DNA from condensed and dispersed human chromatin

Condensed and dispersed chromatin fractions were isolated from human placental nuclei. The DNA of each fraction was purified and characterised by isopycnic centrifugation, thermal fractionation on hydroxylapatite (HAP) and sequence complexity studies. The DNAs had identical buoyant densities in neutral CsCl (1.698 g/cm³) and similar melting profiles on HAP. Analytical ultracentrifugation in Ag⁺-Cs₂SO₄, however, showed that satellite DNAs were present in the condensed fraction DNA (DNA_C) but were not visible in the dispersed fraction DNA (DNA_D). In addition, DNA_C was found to be enriched in highly reiterated sequences (20% reassociated by C₀t 10^?3) which can be correlated with the presence of satellite DNAs, whereas DNA_D contained only 3% of these fast reassociating sequences. In contrast DNA_D contained 30% intermediate sequences (reassociating between C₀t 10^?3 and C₀t 100) which represent only 10% of DNA_C. The reassociated highly repeated sequences of DNA_C showed the presence of two components in both CsCl density gradients and HAP thermal elution studies. This suggests that either there are sequence relationships resulting in partial mismatching between the different highly repeated DNA sequences in this fraction, or that highly repeated sequences are associated with less repetitious DNA. The results are discussed in terms of possible differences in genetic activity between the chromatin fractions.     

Estimation of intracellular pH in muscle of fishes from different thermal environments

A technique based on homogenisation of rapidly frozen tissue was used to investigate the regulation of intracellular pH (pH_i) in freshwater and marine fish from diverse environmental temperatures. The following species were held at ambient temperatures of ca. 1°C (Notothenia coriiceps; Antarctica), 5°C (Pleuronectes platessa, Myoxocephalus scorpius; North Sea), and 26°C (Oreochromis niloticus; African lakes). The effects of seasonal acclimatisation to 4, 11 and 18°C were also examined in rainbow trout in the winter, autumn and summer, respectively. Extracellular (whole blood) pH (pH_e) did not follow the constant relative alkalinity relationship, where pH⁺=pOH⁻ for any particular temperature, over a range of 1–26°C (overall δpH_e/δT=0.009±0.002 U °C⁻¹; P<0.001), apparently being regulated by ionic fluxes and ventilation. Intracellular pH (pH_i) was also regulated independently of pN(=0.5 pK water) in all species of fish examined. The inverse relationship between pH_i and environmental temperature gave an overall δpH_i/δT of −0.010±0.001 U °C⁻¹ (for both white and red muscle) and −0.004±0.003 U °C⁻¹ (cardiac muscle). However, between 1 and 11°C δpH_i/δT was much higher (P<0.001), −0.022±0.003 U °C⁻¹ (white muscle) and −0.022±0.004 U °C⁻¹ (red muscle). The possible adaptive roles for these different acid–base responses to environmental temperature variation among tissues and species, and the potential difficulties of estimating pH_i, are discussed.     

Characterization of segmented double-helical RNA from bacteriophage phi6

The nucleic acid component of bacteriophage φ6 is characterized as a double stranded RNA molecule with a buoyant density of 1.605 g/cm³ and nucleotide composition of C, 27.3%; A, 21.8%; G, 28.9%; and U, 22.0%. The hyperchromicity profile in 0.1 × SSC (SSC is 0.15 m-NaCl, 0.015 m-sodium citrate) demonstrated a rapid increase with a T_m value of 91 °C. The nucleic acid was resistant to degradation by DNase, spleen phosphodiesterase and pancreatic RNase in 2 × SSC buffer but sensitive to degradation by venom phosphodiesterase, pancreatic RNase in 0.01 × SSC and hydrolysis in KOH. Three distinct double stranded RNA species of 2.2, 2.8 and 4.5 × 10⁶ daltons were observed after rate zonal centrifugation, polyacrylamide gel electrophoresis and electron microscopy. This communication therefore presents data establishing a new class of double stranded RNA bacteriophage.     

Isolation and characterization of microbodies and symphyomicrobodies with different buoyant densities from the fungus Blastocladiella emersonii.

Microbodies isolated from sporangia of the aquatic fungus $\text{Blastocladiella emersonii}$ have a mean buoyant density of 1.222 g/cm³ after centrifugation through a linear sucrose gradient, and contain catalase, isocitrate lyase and malate synthase. Microbodies fuse to produce one symphyomicrobody per zoospore at the time of sporogenesis. An increase in density accompanies this process. The symphyomicrobody has a mean buoyant density of 1.292 g/cm³ while the spore's single mitochondrion has a buoyant density of 1.219 g/cm³. Statistical data are also provided for both starting levels and purification of symphyomicrobody and mitochondrial enzyme markers.     

Volume to density relation in adult human bone tissue

Uniformity of tissue mineralisation is a strongly debated issue, due to its relation with bone mechanical behaviour. Bone mineral density (BMD) is measured in the clinical practice and is applied in computational application to derive material proprieties of bone tissue. However, BMD cannot identify if the variation in bone density is related to a modification of tissue mineral density (TMD), a change in bone volume or a combination of the two. This study was aimed to investigate whether TMD can be assumed as a constant in adult human bone (trabecular and cortical).A total number of 115 cylindrical bone specimens were collected. An inter-site analysis (96 specimens, 2 donors) was performed on cortical and trabecular specimens extracted from different anatomical sites. An intra-site study (19 specimens, 19 donors) was performed on specimens extracted from femoral heads. Bone volume fraction (BV/TV) was computed by means of a micro-computed tomography. Furthermore, ash density (ρ_ash) was measured. TMD was computed as the ratio between ρ_ash and BV/TV.It was found that the TMD of trabecular (1.24±0.16 g/cm³) and cortical (1.19±0.06 g/cm³) bone were not statistically different (p=0.31). Furthermore, the linear regression between ρ_ash and BV/TV was statistically significant (r²=0.99, p<0.001). Intra- and inter-site analyses demonstrated that the mineral distribution was independent of the extraction site.The present study suggests that TMD can be assumed reasonably constant in non-pathological adult bone tissue. Consequently, it is suggested that TMD can be managed as a constant in computational models, varying only BV in relation to clinical densitometric analysis.     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Buoyant density centrifugation of sea urchin embryo chromatin on sucrose-glucose gradient

Unsheard chromatin isolated from sea urchin embryos was submitted to buoyant density centrifugation in sucrose-glucose gradients. The main peak of blastula chromatin was at a density position of 1.299±0.028±0.009 g ml^-1 whereas at gastrula stage a shift to a lower buoyant density position of (1.276±0.021±0.007 g ml^-1) was observed. Besides the main peak, a small band with a density of 1.18 g ml^-1 was noticed. The lighter fraction differed from the heavy one in a higher histone to DNA ratio, a lower proportion of the F-1 histone, and a lower nonhistone to DNA ratio. The most pronounced developmental alterations of proteins were observed at the level of nonhistone protein patterns of the light fractions.     

Crystallographic data of the selenoenzyme glutathione peroxidase

Glutathione peroxidase prepared from bovine erythrocytes yields small, but well-ordered plate-like crystals. X-ray investigation shows them to belong to monoclinic space group C2. Unit cell dimensions are: $\text{a = 90.4}\text{A}\text{?}\text{, b = 109.5}\text{A}\text{?}\text{, c = 58.6}\text{A}\text{?}$, β = 99 ° ± 15 min. The crystal density is ?_c = 1.36 ± 0.02 g.cm^?3. Consequently, the asymmetric unit of the crystal cell is occupied by one tetrameric molecule of M_r 84,000. Matthew's (1968) parameter Γ_M is calculated to be 1.71 ų/dalton.     

Nuclear DNA in normal and refed Amoeba proteus

Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm³ with a satellite at 1.714 g/cm³ which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm³. When cells are starved and re-fed with food labeled with [³H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes.     

Characterization of entomopox virions of the army cutworm,Euxoa auxiliaris (Lepidoptera: Noctuidae)

Virions were released from virus-containing inclusions (VCI) of an entomopoxvirus of the army cutworm, Euxoa auxiliaris, with carbonate-thioglycolate solution. Knoblike projections present on the surface of the viral envelope were removed by digestion with trypsin. Trypsin-treated virions were homogeneous in both sucrose and CsCl gradients. The virions were similar to vertebrate poxviruses in morphology, contained 1.13 ± 0.3% DNA and had a buoyant density of 1.261 ± 0.003 gm/cm³ in CsCl. The virion preparations were infective and possessed RNA polymerase activity. Of eight species of Lepidoptera tested, only the species from which the virus was originally isolated proved susceptible to infection.     

Particle properties of parsnip yellow fleck virus

Particle preparations of parsnip yellow fleck virus (PYFV) isolates A-421 and P-121, representing the two major serotypes, were made by clarifying leal extracts with ether or butan-1-ol and concentrating the virus particles by precipitation with polyethylene glycol and differential centrifugation. The preparations contained c. 31 nm-diameter particles comprising two sedimenting components. Top component (T) consisted of stain-penetrable protein shells with A₂₆₀/A₂₈₀= 0.8–0.9, sedimentation coefficient (S₂₀) = 56 S (A-421) or 60 S (P-121), and buoyant density = 1.297 g/cm³. Bottom component (B) consisted of nucleoprotein particles, not penetrable by negative stain, with A₂₆₀/A₂₈₀= 1.9, sedimentation coefficient (S⁰_20.w) = 148 S (A-421) or 153 S (P-121), and buoyant density = 1.520 g/cm³ (A-421) or 1.490 g/cm³ (P-121). Yields of B component particles were up to c. 1 mg/100 g leaf tissue (both isolates); yields of T component particles were up to c. 0.6 mg (A-421) or 5.5 mg (P-121) per 100 g leaf tissue. PYFV particles were found to contain a single RNA species (mol. wt c. 3.4 × 10⁶, c. 9800 nucleotides), constituting 40% of the particle weight, and three polypeptide species, of mol. wt (× 10 ³) 30 , 26 and 24 (A-421) or 31 , 26 and 23 (P-121).     

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 and nuclear genomes in the colorless alga Polytoma

DNA isolated from purified nuclei of Polytoma obtusum has a buoyant density of 1.711 g/ml in CsCl, a Tm of 91.3° C in SSC, and a G + C content of 52.5% as determined by base composition analysis. Thermal dissociation and reassociation studies indicated that this nuclear DNA contains a considerable amount of heterogeneity. Under appropriate reannealing conditions for denatured DNA, about 15% of the DNA reannealed to form a satellite peak at a density of 1.711 g/ml within one hour. Native DNA fractions of different average buoyant densities, ranging from 1.723 to 1.708 g/ml were also obtained in a preparative CsCl gradient, indicating the presence of intermolecular heterogeneity at a molecular size of 8.5×10⁶ daltons. The nuclear DNA reassociated as three distinct classes. The very fast species constituted about 20 % of the total hyperchromicity, the class of intermediate rate comprised roughly 10% of the nuclear DNA, while the remaining 70% consisted of unique sequences. The haploid genome set was estimated by renaturation kinetics studies to contain 5.0×10¹⁰ daltons of DNA or 7.5×10⁷ nucleotide pairs. The analytical complexity of the total nuclear genome was found to be 9.35×10¹⁰ daltons, thus indicating that vegetative cells of P. obtusum are diploid.