Properties of satellite DNA from Brassica nigra

The DNA components of B. nigra were preparatively separated by equilibrium ultracentrifugation in a CsCl density gradient, the buoyant density of the main component being 1,696 g . cm-3, that of the satellite component--1,704 g . cm-3. The properties of individual DNA fractions were investigated. Four major components could be observed on the differential melting curve of satellite DNA. Using the reassociation kinetics method it was shown that 30% of satellite DNA are presented as a fast reassociating component with a length of a repeated unit of approximately 2,5 . 10(3) nucleotide pairs. The calculated values of Tm and buoyant density suggest that the m5C content in satellite DNA is lower than that in the main component. During equilibrium ultracentrifugation in the density gradients of actinomycin D--CsCl and Hg2+--Cs2SO4 the satellite DNA is split into 4 major components.     

An A + T-rich satelitte DNA in a monocotyledonous plant, Cymbidium.

The DNA of aseptically grown protocorms of a Cymbidium hybrid and in vitro developed leaves, as well as DNA of leaves and flower buds of Cymbidium ceres from the greenhouse, was analysed by analytical ultracentrifugation and thermal denaturation. Upon ultracentrifugation a satellite DNA with a buoyant density of 1.682 g/cm-3 appears as a shoulder on the main band (density 1.694 g/cm-3). Thermal denaturation reveals an inhomogeneous main peak with the major component melting at 84 degrees C and a separate peak melting at 75 degrees C. This is the first demonstration of a satellite DNA in a monocot, and one of the rare examples of a major A + T-rich DNA fraction in a plant.     

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.     

Unusual properties of the DNA from Xanthomonas phage XP-12 in which 5-methylcytosine completely replaces cytosine.

Xanthomonas phage XP-12 contains 5-methylcytosine completely replacing cytosine. This substitution confers several unusual properties upon XP-12 DNA. The buoyant density of XP-12 DNA in CsCl gradients is 1.710 g/cm-3, 0.16 g/cm-3 lower than that expected for a normal DNA with the same percentage of adenine plus thymine. The melting temperature for XP-12 DNA in 0.012 M Na+ is the highest reported for any naturally occurring DNA, 83.2 degrees C, 6.1 degrees C higher than that of normal DNAs with the same percentage of adenine plus thymine. Unlike the minor amounts of 5-methylcytosine found in most plant and animal DNAs, the 5-methylcytosine residues of XP-12 derive their methyl group from the 3-carbon of serine instead of from the thiomethyl carbon of methionine. .     

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.     

The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu

Mitochondrial DNA from Ustilago cynodontis has been investigated in several of its properties. Its dG + dC content is equal to 33.5%; its buoyant density (1.698 g/cm3) is higher, by 5 mg/cm3, and its melting temperature (82.5 degrees C) is lower than expected for a bacterial DNA having the same base composition; the first derivative of its melting curve indicates a large compositional heterogeneity, its molarity of elution from hydroxyapatite is high, 0.28 M phosphate, and allows its partial separation from nuclear DNA. Degradation by micrococcal nuclease indicates that about 25% of the DNA is formed by stretches having no more than 15% dG + dC. Finally, the unit size of mitochondrial genome is about 50 X 10(6). In most of its properties, the mitochondrial genome of U. cynodontis presents strong analogies with that of Saccharomyces cerevisiae. A parallel investigation on mitochondrial DNA from Acanthamoeba castellanii which has as genome unit size of only 27 X 10(6), has shown that this shares with the former the dG + dC content (32.9%), the melting temperature (82.5 degrees C), a large compositional heterogeneity and a very similar pattern of micrococcal nuclease degradation; its buoyant density (1.692 g/cm3) and its molarity of elution from hydroxyapatite (0.25 M phosphate) are, however, normal, probably because of a different short-sequence pattern and the fact that its dA + dT-rich stretches are shorter, on the average.     

Nucleotide Composition of Nuclear and Mitochondrial Deoxyribonucleic Acid of Fungi

The buoyant density of nuclear and mitochondrial deoxyribonucleic acid (DNA) from 14 species of fungi was determined by CsCl density gradient equilibrium centrifugation. The buoyant density of both types of DNA was the same for all three Mucorales analyzed. The buoyant density of mitochondrial DNA was significantly lower than that of the nuclear DNA for nine species of Ascomycetes and two species of Basidiomycetes. No simple correlation could be obtained from the comparison of the two types of DNA. Mitochondrial DNA represented a very small proportion of total DNA. Heat-denatured mitochondrial DNA renatured more readily than nuclear DNA.     

Characterization of the molecular components in kinetoplast- mitochondrial DNA of Trypanosoma equiperdum. Comparative study of the dyskinetoplastic and wild strains

The structure of the kinetoplast DNA of Trypanosoma equiperdum has been studied and compared to the structure of the circular mitochondrial DNA extracted from a dyskinetoplastic strain of T. equiperdum. In T. equiperdum wild type, the kinetoplast DNA constitutes approximately 6% of the total cellular DNA and is composed of approximately 3,000 supercoiled minicircles of 6.4 x 10(5) daltons and approximately 50 circular supercoiled molecules of 15.4 x 10(6) daltons topologically interlocked; The buoyant density in CsCl of the minicircles is 1.691 g/cm 3. The large circles have a buoyant density of 1.684 g/cm 3, are homogeneous in size and are selectively cleaved by several restriction endonucleases which do not cleave the minicircles. The cleavage sites of six different restriction endonucleases have been mapped on the large circle. The minicircles are cleaved by two other restriction endonucleases, and their cleavage sites have been mapped. The mitochondrial DNA extracted from the dyskinetoplastic strain of T. equiperdum represents 7% of the total DNA of the cell and is composed of supercoiled circles, heterogeneous in size, and topologically associated in catenated oligomers. Its buoyant density in CsCl is 1.688 g/cm 3. These molecules are not cleaved by any of the eight restriction endonucleases tested. The reassociation kinetics of in vitro labeled kDNA minicircles and large circles has been studied. The results indicate that the minicircles as well as the large circles are homogeneous in sequence and that the circular DNA of the dyskinetoplastic strain has no sequence in common with the kDNA of the wild strain.     

Effect of incubation conditions, inhibitors, 2-mercaptoethanol and testosterone on RNA synthesis in ram spermatozoa

In vitro studies on RNA synthesis using washed ram spermatozoa were carried out by measuring the incorporation of (3)H-uridine into RNA. Penicillin-G (100 mug/ml medium) was added to prevent contamination by microorganisms. Spermatozoa were quickly separated from seminal plasma by washing twice in Tris-HCl buffer (at pH 7.2) and centrifuged at 1,000 g for 5 min. Washed spermatozoa were then diluted to 1 10 , 1 20 or 1 40 (v/v) by the same buffer system (containing 400 mg% glucose) and were incubated in air at 37 degrees C for 1, 2 and 4 h. Results indicated that the rate of RNA synthesis was maximal at 1 40 semenbuffer dilution (5-8 x 10(7) spermatozoa/ml) and increased linearly up to 4 h of incubation. The rate of RNA synthesis at 1 40 dilution also increased linearly as the dose of exogenous glucose substrate was increased up to 400 mg%. Denaturation of the ram spermatozoa by 1% HgCl(2) caused almost complete inhibition of RNA synthesis that amounted to 97% of the control samples. Incubation of spermatozoa with 50, 100 or 200 mug/ml chloramphenicol also inhibited uridine incorporation by 86 to 94%, while equivalent doses of cycloheximide did not. On the other hand, the incorporation of (3)H-uridine into the RNA of ram spermatozoa was significantly enhanced by graded doses of 2-mercaptoethanol (0.2, 0.4 and 0.8 muM) and of testosterone (15 and 30 mug/ml). The results of this study indicate RNA synthesis, mainly of mitochondrial origin, by mature ram sperm. The data also suggest a role for intracellular cyclic adenosine monophosphate in the regulation of sperm RNA synthesis.     

Cyclization of three satellite components of calf thymus DNA.

Cyclization of denatured and reannealed satellite components of calf thymus DNA was studied by electron microscopy. All three satellite DNA components studied (1.707g/cm-3, 1.714g/cm-3 and 1.721g/cm-3) form circular structures indicating that the sequences of the calf thymus satellite DNAs are arranged in a tandemly repetitious manner. Under appropriate annealing conditions the amount of circular structures is reproducible and practically no aggregates are formed. By comparison of cyclization experiments under defined conditions it is demonstrated that individual satellite components differ in the amount of circular structures formed during reassociation and in the distribution of linear and circular molecules. From the distribution of the contour lengths of circular molecules we conclude that the length of the repetitive sequence decreases with increasing buoyant density of the satellite components. The average lengths of the repetitive sequences calculated from electron microscopy measurements are in good agreement with those from renaturation kinetics.     

MP13, a generalized transducing bacteriophage for Bacillus megaterium.

The first generalized transducing bacteriophage reported for Bacillus megaterium has been characterized. Optimum conditions for lysate production and transduction procedures were established so that transducing frequencies of 8 x 10(-6) and higher are now possible. The phage, MP13, has a head diameter of 97 nm and a contractile tail (202 by 17 nm) and adsorbs to the periphery of the cell. MP13 was inactivated rapidly at 60 degrees C, but not at 55 degrees C, and was sensitive to toluene, ether, and chloroform. When centrifuged in a neutral CsCl gradient, two bands were observed, a major band of 1.490 g cm-3 and a minor band of 1.482 g cm-3 buoyant density. The major band contained only infective particles, whereas the minor band contained both infective and transducing particles. Phage DNA was resistant to several restriction endonucleases, but yielded 9 fragments with MboI, more than 34 with HindIII, and 7 with BstEII. The molecular weights for the fragments from MboI-BstEII double digests total 97 x 10(9).     

Structure and synthesis of a lipid-containing bacteriophage. The molecular weight and other physical properties of bacterophage PM2.

Several physical and chemical parameters of bacteriophage PM2 have been measured. The sedimentation constant was determined to be s-20,w=293 S. The buoyant density in sucrose at 20 degrees C was 1.24 g cm+-3 and in CsCl at 25 degrees C was 1.29 g cm-3. The high-speed equilibrium centrifugation method of Yphantis (1964) was used to measure the molecular weight of PM2. The necessary auxiliary parameters were also determined. A value of 0.771 plus or minus 0.005 cm-3 g-1 for the apparent specific volume at constant chemical potential in 1 M sodiium chloride has been obtained by pycnometry; the viral concentration was determined using the absorption coefficient at 260 nm (4.60 plus or minus 0.10 cm-2 mg-1), which in turn was calculated from the phosphorous content of the virus (17.89 plus or minus 0.28 mu-g of P per mg dry weight dry weight of virus). The molecular weight of PM2 determined with these parameters is (44.1 plus or minus 1.2 x 10-6). From the phosphorous content of the virus, the percentage of phosphorous known to be in its DNA (Camerini-Otero and Franklin, 1972), and the molecular weight of the bacteriophage, we have calculated a molecular weight for PM2 DNA of 6.26 x 10-6, which confirms values determined using empirical relationships.     

The significance of promitochondrial structures in rat liver for mitochondrial biogenesis

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4-5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3-4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm(3). 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.     

Purification of the JS-3 isolate of Herpesvirus ovis (Bovid herpesvirus 4) and some properties of its DNA.

A procedure incorporating the use of heparin was developed to purify Herpesvirus ovis. The viral DNA has a buoyant density of 1.706 +/- 0.001 g/cm3, and the sedimentation constant was estimated to be 47.5 +/- 1.5S; from the latter, the molecular weight was calculated as 67.3 +/- 5.4 X 10(6). Estimates of the guanine-plus-cytosine content made from the buoyant density and melting point (72 degrees C) gave levels of 47 and 46%, respectively.     

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.     

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.     

Effect of incubation conditions, inhibitors and seminal plasma on protein synthesis in ram spermatozoa

The effects of incubation time (15 min-4 h), rate of semen to buffer dilution (1/10-1/40), and concentration of glucose (5.5-22 mM) on the rate of protein synthesis by ejaculated washed ram spermatozoa were determined. The rate of protein synthesis increased linearly as incubation time, dilution rate, and the glucose concentration increased. Denaturation of sperm protein with 1% HgCl2 caused an almost complete inhibition of amino acid incorporation. Protein synthesis over a period of 4 h was also inhibited by chloramphenicol but was not affected by cycloheximide. Protein synthesis and uptake of [14C]cAMP by washed ram spermatozoa was also significantly inhibited by the inclusion of 2-8% seminal plasma in the buffer. The present results indicate that the authentic protein synthesis by mature ram spermatozoa is mainly of mitochondrial origin. The data also suggest a role for intracellular cAMP in the regulation of sperm protein synthetic activity.     

STUDIES ON MITOCHONDRIA : II. Mitochondrial DNA of Thoracic Muscles of Schistocerca gregaria

The buoyant densities of the nuclear and mitochondrial DNA from the thoracic muscles of Schistocerca gregaria were found to be 1 702 and 1.689 g/cm³, respectively, corresponding to guanine plus cytosine (G + C) content of 42.2 and 30% A preliminary treatment of the mitochondrial pellet with DNase (25°C, 20 min) is necessary to eliminate the contaminating nuclear DNA. The mitochondrial DNA renatures readily after heat denaturation and incubation at 65°C. The DNA released from the mitochondrial pellet by osmotic shock consists of circular open and closed molecules with a contour length around 5 µ The instability of insect mitochondria in in vitro preparations is discussed.     

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.     

The genome of Theileria parva: some structural properties

The DNA of the protozoan Theileria parva, the causal agent of the bovine East Coast Fever, has been prepared at least 99% pure from the intra-erythrocytic form of the parasite. Its buoyant density was found to be 1.696 g/cm3 and its calculated G + C content was 36.7%. Fragmentation of T. parva by the restriction enzyme EcoRI provides some evidence of the presence of repetitive DNA sequences.