Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CsCl

When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm³. Infectious EEE virus banded in two positions; most of the virus banded at 1.20 g/cm³ and a lesser amount banded at 1.22 to 1.23 g/cm³. Analysis of radioactive profiles of CsCl-fractionated EEE virus labeled with either ³²PO₄ or ³H-uridine suggested that the hemagglutinin was stripped from the intact EEE virion. The viral origin of the hemagglutinin was verified by inhibition with specific antiserum. Attempts to differentiate between infectious EEE virus of the different buoyant densities showed that the denser particle was neither a virus contaminant nor a density mutant. No evidence was obtained to indicate that the denser particle was an immature form of EEE virus. The two infectious EEE species obtained after CsCl fractionation were indistinguishable antigenically. Furthermore, unfractionated as well as CsCl-fractionated EEE virus sedimented at about 260S in sucrose gradients. These results together with the results of rebanding experiments suggested that the denser EEE species (1.23 g/cm³) results from a salt (CsCl)-induced alteration or breakdown of the EEE virion (1.20 g/cm³), and that it arises as the hemagglutinin is stripped from the surface of the EEE virion.     

Location of Satellite and Homogeneous DNA Sequences on Human Chromosomes

HUMAN DNA^1,2 contains at least two satellite fractions—satellite I (0.5% of the genome) which bands at a density of 1.687 g/cm³ in neutral CsCl and satellite II (2% of the genome) which bands at 1.693 g/cm³. The main band DNA has an average buoyant density between 1.670 and 1.720 g/cm³ and a light shoulder (constituting 15% of the genome) with a buoyant density of 1.696 g/cm³, referred to as homogeneous mainband. Satellite DNA has been observed in many higher organisms³, usually with an unknown function, notable exceptions being cistrons coding for ribosomal RNA⁴ and the DNA coding for histone messenger RNA⁵. To investigate possible functions of human repetitive DNA we have studied the annealing of complementary RNA fractions to chromosomes using in situ hybridization^6,7. We describe here preliminary observations using human satellite II and homogeneous mainband fractions.     

Separation of B and C Type Virions by Centrifugation in Gentle Density Gradients

Murine type B particles were separated from type C (Rauscher leukemia virus) by means of gentle (low-increment rate) density gradients. The best separation was obtained when the density ranged from 1.13 to 1.20 g/cm³ when sucrose was used and from 1.12 to 1.28 g/cm³ with CsCl. The buoyant densities of the B and C particle bands in sucrose were 1.18 and 1.16 g/cm³, respectively. The CsCl gradient gave a better separation with the B particles banding at a density of 1.20 g/cm³ and with the C particle density little different from its value in sucrose.     

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.     

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.     

Role of lysine in the replication of reovirus: I. Synthesis of complete and empty virions

Lysine is essential for the replication of infectious reovirus. Omission of lysine from the extracellular medium not only permitted the continued synthesis of structural viral proteins and viral double-stranded ribonucleic acid (RNA), but also caused an enhanced formation of viral structures which were separable by isopycnic sedimentation of CsCl into a top band consisting of empty particles with a buoyant density of 1.29 g/cm³ and essentially free of viral RNA, and two lower bands which were difficult to resolve and had an average buoyant density of 1.37 g/cm³. The lower bands contained most of the viral nucleic acid. The above effects were reversed when lysine was restored early after infection. In contrast, a single band with a buoyant density of 1.38 g/cm³ was obtained from lysine-plus infected cells.     

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.     

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.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

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.     

Fractionation of somatic and sperm chromatin during spermatogenesis in fish

A new approach is suggested for studying changes in the interactions of protein with DNA in the cells. Measurements of the buoyant density of chromatin were performed in somatic cells and in cells undergoing meiosis in fish. During the process of spermatogenesis some of the somatic histones on the DNA are replaced by a new class of proteins; consequently, the mature sperm contains a unique type of protein having a low mol. wt and a high proportion of arginine.The chromatin obtained from mature sperm is composed of a single component with a density of 1.48–1.49 g/cm³ as measured by CsCl equilibrium sedimentation. On the other hand, somatic cells contain chromatin with lower densities. Chromatin obtained from erythrocytes contains a single component with a density of 1.41–1.42 g/cm³ while liver chromatin shows two components; a main component with a density of 1.45–1.46 and a more heterogeneous component with a lighter density (1.32–1.35). There is a correlation between the buoyant density of the chromatin, the type of its basic proteins and the level of biosynthetic activity in the cells.     

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.     

Buoyant densities of DNA of mammals

One characteristic of DNA, CsCl buoyant density peak values, was determined for DNA samples isolated from 93 species belonging to 11 orders of mammals. The CsCl buoyant density values varied over a very narrow range, 1.696–1.701 g/cm³. Satellite DNAs were found in a number of species. The function and origin of these satellite DNAs are not known.This work was supported by grant DRG-269 from the Damon Runyon Memorial Fund for Cancer Research, Inc., and GB-6657 from the National Science Foundation.     

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.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Effect of L-phenylalanine on I-methyladenine production and spontaneous oocyte maturation in starfish

5-Bromodeoxyuridine (BUdR)-resistant cells were obtained from N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-treated soybean protoplasts and cultured in liquid nutrient medium containing BUdR (20 μg/ml) and uridine (100 μg/ml). Addition of uridine to the medium improved growth of the BUdR-resistant cells. The growth of BUdR-resistant cells was partly inhibited when hypoxanthine, aminopterine, glycine and thymidine were added to the medium. Both BUdR-resistant and BUdR-sensitive cells exhibited thymidine kinase activity. CsCl density gradient analyses showed that the DNA of BUdR-resistant cells, which were cultured in the presence of BUdR, had a buoyant density of 1.703 g/ml, while the DNA of the parental soybean cells grown without BUdR had a buoyant density of 1.692 g/ml. Uptake of ³H-thymidine or ¹⁴C-BUdR by the cells occurred in both BUdR-resistant and BUdR-sensitive cells. CsCl density gradient patterns of labelled DNA also demonstrated that ¹⁴C-BUdR and ³H-thymidine were incorporated into the DNA of BUdR-resistant cells, as well as into that of BUdR-sensitive cells.     

Isolation and composition of bacteriophage-like particles from kappa of killer Paramecia

Summary Phage-like particles from kappa of stock 562 of Paramecium aurelia have been isolated by CsCl density gradient centrifugation. Analyses show that the particles contain about 1.6×10¹⁶g DNA and 2.0×10^-16g protein. Their buoyant density is approximately 1.47. DNA from the particles has a buoyant density very close to that of whole kappa DNA. The presence of DNA in the particles has been confirmed by a cytochemical technique. The results support the conclusion that kappa contains a bacteriophage.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.     

Bacteriophage-P22 derivative with high density

Bacteriophage P22Cm21 was differentiated in some characters from the original phage P22. The buoyant density in CsCl solution of phage P22Cm21 was higher than that of phage P22 by as much as 0.007 g per cm³. The possible biological implication involved in this higher density is discussed.     

Melon rugose mosaic virus, the cause of a disease of watermelon and sweet melon

A mechanically transmissible virus with isometric particles c. 32 nm in diameter, was isolated from infected watermelons and sweet melons in the People's Democratic Republic of Yemen. Purified virus preparations contained two major sedimenting components with sedimentation coefficients of 61S and 117S. In isopycn ic centrifugation in CsCl the particles formed a single band of buoyant density 1.39 g cm^-3. Preparations of virus particles comprised of a single polypeptide of mol. wt c. 22 000 and ssRNA of mol. wt 2.1 × 10⁶. The virus was serologically related to three of six subgroups of tymoviruses tested. The name melon rugose mosaic virus is proposed for this newly described virus.