Molecular structure of adeno-associated virus variant DNA

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.     

Rat hepatoma cells nucleolar DNA. 2. Analysis of nucleolar DNA.

The analysis by CsCl density gradient of nucleolar DNA has revealed that the 1.700 g/cm3 main component can be subdivided in three subcomponents with buoyant densities of 1.707 g/cm3, 1.700 g/cm3, 1.690 g/cm3. The 1.707 g/cm3 and 1.690 g/cm3 components contain all the repetitive sequences which comprise 15 % of the total nucleolar DNA. The ribosomal cistrons are found in components having buoyant density between 1.707 g/cm3 and 1.725 g/gm3. Sodium-p-aminosalicylate-DNA interactions have revealed that only the 1.700 g/cm3 fraction has a destabilized secondary structure. The possible localization of these different fractions on peri and intranucleolar fractions is discussed.     

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.     

Physical and Biological Properties of Dengue-2 Virus and Associated Antigens

Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients. Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface. Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm(3) and from cell culture at 1.24 g/cm(3). During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm(3). It was shown in electron micrographs to consist of virion fragments. A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm(3) in CsCl, and appeared as "doughnut" forms measuring 13.8 to 14 nm in diameter. A third component, with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.     

Association of a murine 53,000-dalton phosphoprotein with simian virus 40 large-T antigen in transformed cells.

Serum raised against a mouse 53,000-dalton (53K) phosphoprotein precipitates both the 53K immunogen and simian virus 40 large-T from lysates of simian virus 40-transformed 3T3 cells. This serum, designated F5, does not recognize antigenic determinants on native or denatured large-T and precipitates large-T because the 53K phosphoprotein forms a stable complex with large-T. This complex sediments at 23S on sucrose density gradients, corresponding to a molecular weight of 600K to 1,000K, and appears to contain only 53K and large-T as major components. It is held together by noncovalent bonds and is located in the cell nucleus. All the 53K immunoprecipitated from cell lysates by F5 is present in the high-molecular-weight complex, but large-T can be separated into a complexed and a free form on sucrose density gradients. The complexed form of large-T is more readily phosphorylated than the free form. We have been unable to detect an association of large-T with comparable host cell proteins during productive infections with simian virus 40.     

Characterization of the genome of the small free-living amoeba Acanthamoeba castellanii.

The cellular DNAs of Acanthamoeba castellanii have been characterized by their behaviour in CsC1 density gradients, by their thermal denaturation and by their renaturation kinetics. Whole-cell DNA exhibits, on CsC1 density gradients, a major peak with a density of 1.717 g/cm3 (major component) and a minor peak with a density of 1.692 g/cm3 (minor component). The major component is nuclear and the minor component is of cytoplasmic origin. The latter contains mitochondrial DNA as well as an extramitochondrial DNA fraction. Reiterated sequences make up approximately 14% of the total and are mainly cytoplasmic. They are characterized by three families of nucleotide sequences. The mitochondrial DNA exhibits a complex renaturation pattern. The fast renaturing component has a calculated complexity of 4.107 daltons. The slower renaturing component has a kinetic complexity tentatively estimated as 1.1010 daltons. The melting profile of mitochondrial DNA suggests heterogeneity in base composition.     

Formaldehyde-induced tight binding of protein to RNA in particles of rod-like virus

The formaldehyde-induced formation of tightly bound RNA-protein complexes of rod-like plant viruses was studied. The preparations of tobacco mosaic virus and closely related cucumber virus 4 were incubated with 1.5% formaldehyde for 20-50 hrs at 50 degrees C. Then the viral particles were disrupted, free protein was removed and viral RNA was centrifuged in the linear gradient of Cs2SO4. The RNAs from the formaldehyde-untreated viruses and RNA from the formaldehyde-treated tobacco masaic virus had the density of 1.65-1.66 g/cm3, while RNA from the formaldehyde-treated cucumber virus had the density of 1.57-1.42 g/cm3, depending on the incubation time. This is indicative of the protein binding to RNA. Treatment of the cucumber virus complex with pronase resulted in a liberation of free RNA with the density of 1.66 g/cm3; incubation for 2 min at 100 degrees C in a dissociating mixture (2% sodium dodecyl sulfate + 0.2% mercaptoethanol) did not cause the dissociation of the complex. Polyacrylamide gel electrophoresis showed that the most part of the protein molecules are bound within the complex not by covalent protein-protein cross-links.     

Molecular weight of detergent-solubilized prostaglandin-F2 alpha receptor from bovine corpora lutea

A prostaglandin F2 alpha receptor localized in plasma membranes of bovine corpus luteum cells was solubilized by treatment with Triton X-100. Sepharose chromatographies of ([3H]prostaglandin F2 alpha)-receptor complex gave a Stokes' radius of 630 nm. In the absence of detergent, aggregated forms of the receptor appeared. Sedimentation experiments of solubilized receptor in sucrose/H2O and sucrose/2H2O density gradients gave the following values: sedimentation coefficient (S20, w) 4.6 S; partial specific volume (VB) 0.78 cm3/g and frictional ratio (f/fo) 1.6. Based on the sedimentation coefficient and the Stokes' radius and assuming that the receptor is a non-glycosylated protein the molar mass of the receptor-(Triton X-100) complex was 144000 g/mol. The VB value indicated that ca. 26% of the weight represented bound detergent and that the molecular weight of the prostaglandin F2 alpha receptor is approximately 107000.     

A physical study of the ovine genome

The ovine genome has been divided into some seventy-five fractions using 3,6-bis(acetatomercurimethyl)dioxane (BAMD) in conjunction with Cs2SO4 density-gradient-equilibrium centrifugation. Distinct macromolecular populations detected have buoyant densities in CsCl of 1.700, 1.707, 1.714, 1.716, 1.717, 1.721, 1.724 and 1.725 g/cm3. The 1.724 g/cm3 material appears in a number of non-contiguous fractions obtained from BAMD-Cs2SO4 centrifugation suggesting its presence at a number of different sites in the genome. Within two regions of buoyant density (1.701 g/cm3 to 1.707 g/cm3 and 1.708 g/cm3 to 1.717 g/cm3) the analyses were unable to resolve discrete populations.     

Ribonucleoproteins containing mRNA in the cytoplasm of mouse plasmacytoma cells]

The rapidly labelled postribosomal ribonucleoprotein (RNP) found in the cytoplasm of mouse plasmacytoma cells were investigated. It has been shown that 45S and 80S particles contain relatively high molecular weight (approximately 12-17S) pulse-labelled RNA similar to the polyribosomal mRNA. No other postribosomal RNP was found which would contain an RNA with similar sedimentation characteristics. In CsC1 density gradients, the postribosomal RNP gives two peaks. One of them, the rapidly labelled component (rho 1.52 g/cm3) is found only in 45S RNP. The other rapidly labelled component (rho 1.36-1.41 g/cm3) is revealed in all investigated regions of sucrose gradients. The latter contains relatively low molecular weight RNA (approximately7-9S). These RNP are supposed to be informosome-like particles. The components with a buoyant density of 1.52 g/cm3 may represent an mRNP-45S subparticles complex. The rapidly labelled mRNA of 80S particles is released after EDTA treatment in the form of mRNP with a buoyant density of 1.45-1.47 g/cm3.     

Complex of DNA with chromatin proteins investigated by isopycnic centrifugation in metrizamide.

Complexes of mouse main band DNA with a fraction of non-histone proteins (NHP), having a high affinity for DNA, in the absence or presence of histones have been investigated by gradient centrifugation in metrizamide. Two types of complexes were formed at an input ratio of NHP to DNA between 1 and 2.5. In metrizamide gradients a majority of DNA was found in the light complex (at the density of 1.14-1.16 g/cm3) even at the very high NHP to DNA ratio. When histones were present in the reaction mixture, most of the DNA was found in the heavy complex (1.19-1.21 g/cm3). The electrophoretic profiles of the proteins recovered from the heavy and light complexes were different; some fractions of nonhistone proteins were present only in the heavy component.     

Alternate forms of the resistance factor R1 in Proteus mirabilis

The resistance factor R1 may exist in either of two stable physical states in Proteus mirabilis PM-1. In one case, the R1 deoxyribonucleic acid (DNA) has a buoyant density of 1.711 g/cm(3) and replicates under stringent control. Cells harboring R1 in this form may transfer drug resistance by conjugation. In the other case, R1 DNA shows two buoyant density classes at 1.707 and 1.714 g/cm(3). The 1.714 g/cm(3) component is replicated under a degree of relaxed control, and strains carrying this form generally cannot transfer drug resistance by conjugation. Intracellular amounts of the R factor-coded enzyme, chloramphenicol acetyltransferase, did not correspond to amounts of plasmid DNA in Proteus, and the enzyme was present in lower amounts than in Escherichia coli. It is proposed that the two states of R1 in Proteus may represent stable associated and dissociated forms of the plasmid.     

Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.     

Density-gradient centrifugation in lithium metatungstate and tris-(hydroxymethyl)aminomethane phosphotungstate

New substances--lithium metatungstate (MTL) and tris-(hydroxymethyl)aminomethane phosphotungstate (PTT)--have been presented for density-gradient preparation. The buoyant densities of protein, RNA, DNA and some nucleoproteins were determined in solutions of these salts. Nucleic acids have been smaller buoyant density (1.1 g/cm3) than the proteins in contrast to CsCl-gradients. The protein in PTT solution have buoyant density 1.5 g/cm3 and in MTL solution 2.0-2,3 g/cm3. It was shown that MTL gradients allow to reach better resolution in nucleoprotein analysis than CsCl gradients.     

Analytical fractionation of cultured hepatoma cells (HTC cells)

Homogenates of HTC cells have been fractionated by differential centrifugation (in four particulate fractions: N, M, L, P, and a supernatant S) or isopycnic banding in linear sucrose gradients. On this basis, the following subcellular organelles may be characterized: (i) Mitochondria, detected by cytochrome oxidase and succinodehydrogenase, are collected in the M and L fractions, and equilibrate, as a narrow band, at a median buoyant density of 1.18 g/cm3. (ii) Lysosomes, detected by the latent hydrolases beta-glycerophosphatase and N-acetyl-beta-glucosaminidase, are largely sedimented in the M and L fractions, and display a broad density distribution pattern with a median value of 1.17 g/cm3. This density is decreased or increased after cultivation of the cells in presence of Triton WR-1339 or Dextran 500, respectively. The behavior of cathepsin D is somewhat at variance with that of the two other hydrolases. (iii) Plasma membrane is tentatively detected by alkaline phosphodiesterase I. Largely recovered in the P fraction, this enzyme equilibrates at a median density close to that of the lysosomal hydrolases; the bulk of cholesterol and about half of the leucyl-2-naphthylamidase are closely associated with alkaline phosphodiesterase I; HTC cells do not contain typical 5'-nucleotidase. (iv) Catalase-bearing particles, of high buoyant density (1.22 g/cm3) are present, but 30-40% of the catalase is also found readily soluble. NADPH- and NADH: cytochrome c reductase, and RNA show more complex distributions. It is suggested that the former enzyme is associated with the endoplasmic reticulum; as in liver, NADH reductase activity is shared between the endoplasmic reticulum and the mitochondria; half of the RNA is associated with free ribosomes of polysomes. True glucose-6-phosphatase could not be detected.     

Hepatitis A antigen isolated from liver and stool: immunologic comparison of antisera prepared in guinea pigs.

Morphologically similar hepatitis A antigen particles (HA Ag)3 have been detected in the stools of patients with type A hepatitis and in the livers of marmosets experimentally infected with hepatitis A virus. To investigate the humoral antibody responses to these antigens and to compare the immunologic properties of HA Ag from these two sources, we immunized guinea pigs with either marmoset liver-derived HA Ag or with human stool-derived HA Ag in complete Freund's adjuvant and measured their antibody responses by immune electron microscopy (IEM) and immune adherence hemagglutination (IAHA). Antibodies reacting with both hepatitis A antigens were elicited in both groups. As determined by IEM, no distinction was seen between the reaction of guinea pig antiserum to each HA Ag tested under code when reacted against either liver-derived or stool-derived HA Ag. Antibodies elicited to marmoset liver-derived HA Ag and human stool-derived HA Ag had similar end point dilution titers by IAHA when tested against either "light" density (1.34 g/cm3) or "heavy" density (1.40 g/cm3) stool-derived HA Ag or liver-derived HA Ag. Low levels of antibody to normal liver or stool control antigens were observed transiently but did not obscure the specific response to HA Ag. These data suggest that morphologically similar HA Ag particles from different sources and with different densities are immunologically similar and may be identical. In contrast to the heterogeneity of surface antigens of hepatitis B virus, the comparable immunogenicity and apparent antigenic homogeneity of HA Ags derived from various sources may simplify the approach to development of a vaccine against viral hepatitis, type A.     

Identification and characterization of a 19-S complex containing a 27 000-Mr protein in Artemia salina.

The cytoplasm of the cryptobiotic Artemia salina gastrula contains a large quantity of a unique 19-S complex. This particle is a specific aggregated form of a 27 000-Mr protein, having a molecular weight of 10(6) and an apparent buoyant density of 1.25 -- 1.26 g/cm3 in sucrose and 1.31 g/cm3 in CsCl. The relative quantity of this 19-S complex decreases significantly with respect to 80-S monoribosomes during the postgastrula development. Biochemical and immunological studies indicate that the 27 000-Mr protein is one of the RNA-binding proteins [Ovchinnikov et al., FEBS Lett. 88, 21 -- 26 (1978)] but is absent in the protein components associated with poly(A)-containing ribonucleoprotein complexes. The possibility is also suggested that the 27 000-Mr protein and Artemia elongation factor eEF-Ts might be related to each other on the basis of amino acid composition and immunological cross-reactivity.     

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.     

Determining particle density distribution of expanded bed adsorbents

This study presents an experimental approach to measure the density distribution of expanded bed adsorption (EBA) matrices. We report on the use of a series of solutions of caesium trifluoroacetate (CsTFA) of varying density spun in a laboratory centrifuge so as to separate representative matrix samples on the basis of bead density. Mass data was used to plot a decumulative density distribution for the matrix. By performing laser light scattering-based measurements on the same samples of matrix the variation in particle size with density was determined. Particle settling velocity distributions were then calculated using these data and compared with a settling velocity distribution calculated on the basis of an assumed constant bead density. The study demonstrates a reliable and simple method for the characterisation of matrix density distribution. For the case of the Streamline matrices tested the particle size distribution is constant with varying bead density. Bead densities varied from 1.5 to 2.1 g/cm3 in the CsTFA solutions. These were then adjusted using bead porosity to give a density range of 1.11-1.33 g/cm3 in aqueous buffer (assumed 1.0 g/cm3) The differences in resultant settling velocity distributions when based upon measured density distribution than when based upon an assumed mean density value were shown to be insignificant. This result confirms experimentally that an assumption of a single constant mean density for EBA particles is acceptable for hydrodynamic modelling and performance prediction purposes.