Characterization of the DNA in DROSOPHILA MELANOGASTER

DNA has been quantitatively extracted from Drosophila melanogaster at various stages of embryonic development and analyzed by isopycnic centrifugation in CsCl and by fractionation on methylated albumin columns. The DNA is composed of three main classes of DNA, as defined by their buoyant density, rho, in CsCl: a bulk DNA, rho = 1.699 g cm(-3), and two satellite DNAs, rho = 1.685 g cm(-3) and rho = 1.669 g cm(-3). These three types of DNA persist throughout the development of the insect. In the unfertilized egg, 80% of the total DNA consists of the satellite DNAs; this amount decreases to 18% during the first three hours after fertilization and then remains constant through embryogenesis. There is a concomitant increase of the satellite DNA's with the bulk DNA after blastoderm formation.     

Satellite DNA in differentiating chick tissues

Embryonic chick DNA from different tissues was examined for differences which might indicate specific DNA amplification in somatic cells. The problem was approached by determining the DNA compositional heterogeneity and searching for possible variation in different tissues of the 12-day chick. Neural retina, muscle, and whole decapitated (general) chick DNA were analyzed in CsCl and Cs2SO4 density gradients. While overloaded CsCl gradients showed a main band (rho = 1.701 g/cm3) and a heavy shoulder (rho = 1.716 g/cm3), overloaded Cs2SO4 gradients displayed a main band (rho = 1.426 g/cm3) and a discrete heavy satellite (rho = 1.447 g/cm3). This satellite, comprising approximately 1% of the whole cell DNA, appeared to be of nuclear origin and not related to mitochondrial DNA, which was found to have a density of 1.426 g/cm3 in Cs2SO4. No differences were found in the densities of the main band or the satellite DNA in the DNA samples isolated from the different tissues. However, the method of DNA isolation was found to be of crucial importance when comparing satellite DNA's among different tissues.     

Satellite Dnas in the Embryos of Various Species of the Genus Drosophila

A tentative evolutionary pattern has been found for two classes of the multiple satellite DNA's found in the genus Drosophila. The satellite DNA's from five Drosophila species (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) were analyzed and found to fall into three arbitrary CsCl buoyant density classes: Class I, rho = 1.661-1.669 g cm(-3), DNA molecules composed of primarily dA and dT moieties; Class II, rho = 1.685 and rho = 1.692, DNA molecules of low GC content; and Class III, rho = 1.711, a DNA of high GC composition. The dAT satellite DNA's appear in all the species studied except D. hydei, the species of most recent evolutionary divergence, whereas the heavy satellite appears only in the two species of most recent divergence, D. virilis and D. hydei.     

Properties of satellite DNA of Phaseolus vulgaris]

Nuclear DNA components of Ph. vulgaris were preparatively separated by equilibrium ultracentrifugation in Hg++-Cs2SO4 density gradient (buoyant density of the major component in CsCl density gradient 1.694 g/cm3., satellite component--1.703 g/cm3). The properties of individual DNA fractions were investigated. The melting curve of satellite DNA of Ph. vulgaris has biphasic character. The observed heterogeneity of satellite DNA component is of intermolecular nature. This is illustrated by the splitting of unsheared satellite DNA into two components during renaturation, as well as by its behaviour in Hg++-Cs2SO4 density gradient at high rf value. The width of satellite DNA reassociation curve covers three decades of Cot. The length of the major repeating sequences of the satellite component is close to the length of phage T2 DNA. During chromatography on MAK column satellite DNA elutes earlier than the major component due to its higher GC-content. It is suggested that one of the satellite DNA fractions of Ph. vulgaris contains rRNA genes.     

Studies on Spinach Chloroplast and Nuclear DNA Using Large-scale Tissue Preparations

A method for the large-scale preparation of spinach chloroplasts using the Spinco Model L-4 zonal ultracentrifuge and for the extraction of DNA from the chloroplasts is described. Thirty-five percent of the chloroplast DNA (rho = 1.706 g/cc) differs from nuclear DNA (rho = 1.695 g/cc) in buoyant density. T(m), base composition, and renaturation properties. Sixty-five percent of the chloroplast DNA (rho = 1.696 g/cc) has the same buoyant density and T(m) as nuclear DNA, but it differs in base composition and renaturation properties.     

Deoxyribonucleic acid in Nitrobacter carboxysomes.

Carboxysomes were isolated from Nitrobacter winogradskyi and Nitrobacter agilis. The icosahedral particles contained double-stranded deoxyribonucleic acid (DNA). In the presence of ethidium bromide and cesium chloride, the particle-bound DNA had a buoyant density of rho 25 = 1.701 g/cm3. Electron microscopy revealed the DNA to be a 14-micron circular molecule.     

Differential replication of satellite and main band DNA during early stages of callus formation in carrot root tissue

DNA synthesis during the early stages of callus formation wasexamined in carrot root slices cultured on an agar medium containing2,4-D. During the first 12 hr of culture, only a low level of3H-thymidine was incorporated into DNA, after which the incorporationrapidly increased and reached a maximum at about 48 hr, thengradually decreased. CsCl density centrifugation of total tissueDNA indicated that a satellite DNA with a buoyant density ofabout 1.712 g/ml replicates in the early phase of the DNA syntheticperiod, then the main band DNA with a buoyant density of 1.695–1.700g/ml starts its replication and continues to be produced throughoutthe synthetic period. The labeled satellite DNA, as well asthe labeled main band DNA, was localized mainly in the subcellularfraction of 1,000 ? g sediments obtained from tissue homogenates.The patterns of cellular localization were not modified by theaddition of Triton X-100 to the initial homogenates. A considerableportion of the labeled satellite DNA was found in nuclease-resistantchromatin subunits after limited digestion of the isolated chromatinwith micrococcal nuclease. (Received August 30, 1979; )     

Ribosomal DNA in spores of Physarum polycephalum

DNA was isolated from plasmodia, spores and newly hatched amoebae of the slime mould Physarum polycephalum. The DNA preparations were fractionated in CsCl gradients and each fraction hybridised to combined 19 S + 26 S rRNA. In all three DNA preparations hybridisation was found to be limited to satellite DNA (rho = 1.714 g/cm3) and at saturation was found to reach a level of 0.16--0.18 % of total DNA. The main band of nuclear DNA (rho = 1.702 g/cm3) did not hybridise appreciably. Further experiments using analytical CsCl gradients revealed that the ratio of satellite to main band DNA was similar in all three preparations. It is concluded that the genes for ribosomal RNA are equally reiterated in spores, hatching amoebae and in plasmodia. They appear to be similarly organised in all stages of the life cycle examined so far.     

Characterization of guinea pig cytomegalovirus DNA.

The genome of guinea pig cytomegalovirus (GPCMV) was analyzed and compared with that of human cytomegalovirus (HCMV). GPCMV and HCMV DNAs were isolated from virions and further purified by CsCl centrifugation. Purified GPCMV DNA sedimented as a single peak in a neutral sucrose gradient and was infectious when transfected into guinea pig embryo fibroblast cells. The cytopathology was characteristic of that seen after infection with GPCMV. Virus DNA purified from virions isolated from infected GPEF or 104C1 cells had a CsCl buoyant density of 1.713 g/cm3, which corresponds to a guanine plus cytosine content of 54.1%. The CsCl buoyant density of GPCMV DNA was slightly less than that of HCMV DNA (1.716 g/cm3), but sufficiently different so that the two virus DNA peaks did not coincide. GPCMV DNA cosedimented with T4 DNA in a neutral sucrose gradient. Restriction endonuclease cleavage of GPCMV or HCMV DNAs with HindIII, XbaI, or EcoRI yielded fragments easily separable by agarose gel electrophoresis and ranging from 1.0 X 10(6) to 25.8 X 10(6) daltons. The number, size, and molarity of GPCMV DNA fragments generated by restriction enzymes were determined. Hybridization of restriction endonuclease-cleaved GPCMV DNA with radioactively labeled HCMV DNA and, conversely, hybridization of restriction endonuclease-cleaved HCMV DNA with radioactively labeled GPCMV DNA indicated sequence homology between the two virus DNAs.     

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 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.     

THE DNA components of the chicken genome.

The organization of the chicken genome was investigated by centrifuging chicken DNA (Mr = 57 X 10(6) in preparative Cs2SO4/Ag+ and Cs2SO4/BAMD density gradients [BAMD = 3.6-bis(acetato-mercurimethyl)dioxane]. An analysis by CsCl density gradient of the DNA fractions obtained from the preparative experiments revealed that 88% of the genome is made up of four DNA components, characterized by buoyant densities of 1.699, 1.702(5), 1.704(5) and 1.708 g/cm3 and representing 39%, 25%, 15%, and 9%, respectively, of the total DNA. The remaining 12% of the genome is formed by seven minor and/or satellite components. The distribution of the ovalbumin gene in a Cs2CO4/BAMD density gradient, as tested with a cloned cDNA probe, coincides with the distribution of the 1.702(5)-g/cm3 component. This shows that the DNA regions flanking the ovalbumin gene are homogeneous in base composition over along distances and that the gene is located on a DNA segment belonging to the 1.702(5)-g/cm3 component.     

Topographic heterogeneity in cholesterol biosynthesis

We have examined the membrane topography of cholesterol biosynthesis in cultured human fibroblasts. We fed the cells with radioacetate and then interrupted the biosynthetic pathway so as to trap labeled intermediates in their subcellular locations. We analyzed homogenates of human fibroblasts labeled biosynthetically from radioacetate by centrifugation to equilibrium on sucrose gradients. The following two methods were used to interrupt cholesterol biosynthesis: incubation at 10 degrees C and treatment with 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, a specific inhibitor of oxidosqualene cyclase. Incubation at 10 degrees C caused the accumulation of radiolanosterol at the expense of cholesterol. The lanosterol appeared predominantly at an unusually buoyant density (20% (w/w) sucrose; d = 1.08 g/cm3) as well as at the density normally labeled at 37 degrees C (30% sucrose; d = 1.13 g/cm3). 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol treatment caused the accumulation of labeled squalene and squalene 2,3-oxide. Reversal of the block permitted the label to progress rapidly as a wave into lanosterol and ultimately into cholesterol. The profiles of the three precursors did not coincide, suggesting that they were mostly in different membranes. Squalene was uniquely confined to a density of 1.18 g/cm3 (40% sucrose) while squalene 2,3-oxide appeared in peaks of density 1.08 g/cm3 and 1.13 g/cm3 (20% and 30% sucrose). Lanosterol was in a peak of density 1.13 g/cm3. Pulse-chase experiments showed that lanosterol synthesized in the membranes at 20% sucrose moved rapidly to the membranes at 30% sucrose where it was converted to cholesterol. The density gradient profiles of the following organelle markers also were monitored: plasma membrane, cholesterol mass; Golgi apparatus, galactosyltransferase; endoplasmic reticulum, RNA, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cytochrome c reductase; peroxisomes, catalase. None of these markers appeared at the buoyant density of 1.08 g/cm3. We conclude that 1) cholesterol biosynthesis may be topographically heterogeneous and 2) newly synthesized squalene 2,3-oxide resides in a buoyant membrane fraction distinct from markers for the major organelles.     

Studies on Resistance Transfer Factor Deoxyribonucleic Acid in Escherichia coli

A variant of the derepressed R factor, R1, which does not contain any of the drug resistance markers, and represents, in large part, the resistance transfer factor (RTF) was studied in Escherichia coli. RTF deoxyribonucleic acid (DNA) was specifically labeled in a female cell after conjugation. Physical characterization of the molecule showed that RTF possessed an average molecular weight of 50 x 10(6) daltons and a buoyant density of 1.709 g/cm(3). By comparison to R1, we calculate that the region of DNA carrying the drug resistance genes is therefore about 20% of the R1 molecule and has a buoyant density of approximately 1.716 g/cm(3). These results support the hypothesis that the single species of R-factor DNA observed in E. coli represents a composite of the 1.709 and 1.716 g/cm(3) replicons seen in Proteus.     

Analysis of eucaryotic DNAs with a restriction endonuclease from H.influenzae: isolation of “hidden” satellite DNAs

The restriction enzymes from H.influenzae have been used to study various eucaryotic DNAs. Fractions resistant to the action of these enzymes have been isolated from rat, mouse and calf DNAs. The method revealed the existence in rat of several components having the properties of satellite DNAs. Mouse DNA was shown to contain a new satellite of buoyant density 1. 704. Therefore, this appears to be a powerfull method for the isolation of "hidden" satellite DNAs.Calf satellite DNAs rho = 1.705 and rho = 1.723 were those resistant to the action of the restriction enzymes whilst satellite DNAs rho = 1.714 and rho = 1.710 gave fragments of discrete lengths, suggesting internal repeat units of 1 500 and 5 000 base pairs respectively.     

Early Replication of Highly Repeated DNA Sequences during Dedifferentiation of Carrot

When carrot explants are placed on an agar medium containingphytohormones, a satellite DNA begins to replicate earlier thanthe bulk DNA during the first cell division cycle. The majorityof this early replicating satellite DNA was previously shownto have an identical buoyant density to ribosomal DNA (rDNA)(Hase et al. 1982). Molecular sizes of EcoRI-digests of ³H-labeledDNA were analyzed in the present study by gel electrophoresisfollowed by fluorography. Most of the labeled DNA bands didnot correspond to EcoRI-digests of carrot rDNA. The resultsindicated that the majority of the early replicating satelliteDNA is not rDNA, but probably a type or types of highly repeatedDNA sequences. (Received June 6, 1985; Accepted November 4, 1985)     

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.     

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.     

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.     

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.