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.     

Properties of Light Particles Produced During Growth of Type 4 Adeno-Associated Satellite Virus

Adeno-associated satellite virus type 4, obtained by repeated undiluted passage, failed to produce distinct bands at the expected density of 1.43 g/cm³ after density gradient centrifugation in CsCl. This phenomenon occurred regardless of the hemagglutinating activity of the starting material. Sharp bands were found at a density of 1.34 to 1.35 g/cm³. These bands contained adenovirions and numerous satellite particles. These latter particles could be distinguished by electron microscopy from standard dense satellite particles by their flattened profiles and deep penetration of negative stains. Dense bands of satellite virus at 1.43 g/cm³ were constantly observed when the inoculum was comprised of highly diluted seed virus. Light satellite particles had a particle to HA ratio comparable with dense particles, but possessed low infectivity. Measurements of contour lengths of extracted deoxyribonucleic acid (DNA) indicate that light particles contain only a small amount of DNA, possibly less than 0.5 × 10⁶ daltons, compared to 1.4 × 10⁶ for the complete satellite DNA molecule.     

Equilibrium centrifugation of measles virus

Measles virus has been centrifuged on different density gradients. It sediments at densities of 1,20 g/cm³ in K-tartrate, of 1,18–1,21 g/cm³ in sucrose, 1,19–1,23 g/cm³ in CsCl and 1,19 g/cm³ in metrizamide gradients. Metrizamide reduced measles virus infectivity. In sucrose gradients sometimes more than one infectious peak was observed. Control Vero cells produced particles of the same densities as measles virus peaks. These peaks did contain actin as the major protein. The relevance of this finding in relation to the presence of actin in measles virus is discussed.     

Identification of viral ribonucleoproteins in the cytoplasm of murine cells chronically infected by a retrovirus.

Ribonucleoproteins were isolated from the cytoplasm of Friend-Eveline cells which produce the Friend virus complex, after a short labelling with [³H] uridine. These particles moved with a sedimentation coefficient of 53S in sucrose gradient and had a buoyant density of 1.46 g/cm³ in CsCl gradient. Analysis of their RNA content showed that they possessed a 35S major species having the size of the viral genome subunit. Moreover, a positive hybridization was observed when RNA of the 53S particles was annealed with viral complementary DNA. No such particles were found in cultures of uninfected murine cells suggesting that 53S RNPs have a viral origin.     

Biophysical Properties of Australia Antigen

Biophysical studies with Australia complement-fixing (CF) antigen showed it to be a particle with a buoyant density of 1.20 g/cm(3) in CsCl, a sedimentation coefficient of 110, and an average diameter of 25 nm. The CF antigen was not inactivated by ether, 1% deoxycholate, 1% Tween 80 or overnight heating at 56 C. The antigen was unstable when treated with 1% sodium dodecyl sulfate. A procedure is described for the isolation and partial purification of Australia antigen from serum by using isopycnic banding and rate separation techniques. Treatment of the 1.20 g/cm(3) Australia antigen with 1% Tween 80 yielded a minor peak of CF activity with a buoyant density of 1.39 g/cm(3) in CsCl.     

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.     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by arginine and biotin: a two-factor analysis

Summary The distribution of glyoxylate-cycle enzymes between microbodies and mitochondria was examined in ethanol-grown Aspergillus tamarii Kita. Particulate activities of catalase and the two glyoxylate by-pass enzymes, malate synthase and isocitrate lyase, were localized in the microbodies. The microbodies had a buoyant density of about 1.23 g cm^-3 after isopycnic centrifugation in linear sucrose gradients. Particulate activities of the other two glyoxycitrate synthase, together with that of succinate dehydrogenase were restricted to the mitochondria, which had a buoyant density of about 1.20 g cm^-3. Catalase also appeared to be localized in a second particle, perhaps the microbody inclusions or the Woronin bodies, having a buoyant density of about 1.26 g cm^-3.     

Messenger ribonucleoprotein particles in silkmoth oogenesis

Silkmoth oocytes contain significant amounts of nontranslating cytoplasmic messenger RNA apparently stored until fertilization. The physical state of this mRNA was examined by bouyant density centrifugation on cesium chloride gradients. Messenger elements were isolated either by oligo(dT) cellulose chromatography or by separation of ooplasm on sucrose gradients. After CsCl density gradient centifugation the mRNA particles banded in a region (1.42–1.48 g/c³) which would indicate a substantial protein content. Electron microscope examination of mRNP fractions revealed particles ranging in size from 180–250 Å.     

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.     

Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K⁺-stimulated Mg²⁺-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557–571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm³. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10–1.11 g/cm³), Golgi (1.12 g/cm³) and mitochondria (1.18 g/cm³). Furthermore, other recent literature indicates that the 1.13 g/cm³ buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K⁺-stimulated Mg²⁺-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm³ density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm³. Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm³ particulate components with minimal contamination by particles of higher densities.     

Biochemistry and ultrastructure of iridescent virus type 29

Physical and chemical parameters of iridescent virus type 29, isolated from the mealworm, Tenebrio molitor, have been analyzed. The icosahedral capsid is 130–135 nm in diameter and is surrounded by a fringe of coarse filaments. The virus has a buoyant density in CsCl of 1.31 g cm^?3 and contains 20 to 25 structural proteins as analyzed by isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The DNA has a buoyant density in CsCl of 1.6874 g cm^?3 indicating a G + C content of approximately 28%. The lipid components of this virus differ from those of the host cell; the virus contains about 80% cardiolipin and 20% phosphatidyl choline.     

The DNAs of the A and B chromosomes of the mealy bug,Pseudococcus obscurus

When the DNA of mealy bugs carrying B chromosomes (+ B:DNA) was compared to the DNA of individuals not possessing Bs (-B:DNA), no significant differences were found using isopycnic centrifugations in CsCl or thermal denaturation analyses. Both DNAs had buoyant densities of 1.693 g/cm³ in neutral CsCl gradients and 1.748 g/cm³ in alkaline CsCl gradients. Satellite DNAs were not detected. The average T_m of +B:DNA was 67.9° C in 0.1 SSC while -B:DNA had an average T_m of 67.4° C in the same solution. However, in situ molecular hybridizations with complementary RNAs (cRNAs) transcribed in vitro from each type of DNA showed considerable differences with regard to the amount of labeling of B chromosomes. Using cRNA to +B:DNA, the average number of silver grains over a B chromosome was 2.1 × the average number of silver grains over individual non-B chromosomes (A chromosomes). In contrast, the ratio (B/A) using cRNA to -B:DNA was less than 0.14. The results are interpreted as meaning that very little DNA is shared in common by both A and B chromosomes.     

Properties of a host range and coat protein variant of broad bean true mosaic comovirus from Vicia faba

Unlike other described isolates of broad bean true mosaic comovirus (BBTMV), a variant, code name SB, infected some non-leguminous plant species and, in N. benthamiana, induced systemic mottling and puckering of the leaves. However, like other described BBTMV isolates, purified SB particle preparations contained isometric particles c. 28 nm in diameter that sedimented as two nucleoprotein components with S_{20, w} values of 90S and 109S; some preparations occasionally contained a component of c. 50S. Virus particles contained two ssRNA species which, when denatured in glyoxal, had estimated M_T values of 2.1 × 10⁶ and 1.3 × 10⁶ and co-electrophoresed with cowpea mosaic virus RNA-1 and RNA-2 respectively. Isolate SB was serologically indistinguishable from British and German isolates of BBTMV. However, SB virus particles contained a major polypeptide (L) of M_r between c. 31 000 and up to three minor ones (S) or M_r between c. 20 000 and 24 000. This contrasts with protein preparations from other BBTMV isolates that typically contain only two polypeptides of M_r c. 37 000 (L) and 21 000 (S). Following isopycnic centrifugation in CsCl, SB particles purified from pea separated into two major components with densities of 1.39 and 1.44 g cm^-3 and a minor component of estimated density 1.43 g cm^-3. In Cs₂SO₄, virus preparations separated into three major components with densities of 1.30, 1.32 and 1.36 g cm^-3 and a minor one of density 1.27 g cm^-3. In CsCl isopycnic gradients, SB particles purified from TV. benthamiana separated into two components with densities of 1.38 and 1.43 g cm^-3. During immuno-electrophoresis in agarose gels, freshly prepared virus and preparations stored for up to 4 days at 4°C contained a single component that migrated rapidly to the anode, whereas similar preparations of an English isolate of BBTMV migrated as a single component that moved only slowly toward the anode but which, within 48 h, contained an additional component with a migration rate similar to that of isolate SB. Isolate SB is therefore a host range variant of BBTMV which, in comparison with previously described isolates of BBTMV, has an increased negative charge of its particles prior to any appreciable degradation of its S protein, and S protein that is degraded less rapidly. These features probably account for the anomalies observed in isopycnic centrifugation.     

Characterization and localization of a virus-like particle in aDrechslera species

A virus-like particle (VLP) has been found in a species of the fungusDrechslera. Four double-stranded RNA species with sizes of 3.8, 2.8, 2.7, and 2.2 kbp were isolated using CF-11 cellulose. These dsRNAs are associated with a 35-nm particle at a density of 1.37 g/cm³ in CsCl. The particle contains a major protein of 117 kDa and a minor protein of 89 kDa. In cellular fractionations of 2-week-old cultures, the VLPs are found in the mitochondrial pellet, but do not band with mitochondria in sucrose step gradients. In 1-week-old cultures, however, the VLP dsRNA is found in the cytoplasmic fraction. VLPs have not been reported previously in this genus.     

Density Gradient Centrifugation of Rubella Virus

Rubella virus was centrifuged in sucrose density gradients. One of two densities could be ascribed to the virus, depending upon the suspending medium used. The virus was found at a density of 1.16 g/cm³ after centrifugation for 18 hr in sucrose gradients prepared in distilled water. By contrast, when the sucrose gradients were prepared in tris(hydroxymethyl)aminomethane (Tris)buffer containing ethylenediaminetetraacetic acid (EDTA), the virus was found at a density of 1.18 g/cm³ after 18 hr of centrifugation. The virus banded at this higher density after only 2 hr of centrifugation when pretreated by overnight incubation in the Tris-EDTA buffer. A kinetic study showed that, in sucrose gradients containing this buffer, the virus gradually migrated as a single peak of infectivity from a density of 1.16 g/cm³ after 2 hr of centrifugation to the higher 1.18 g/cm³ density after 18 hr. The density change was shown to be reversible; after the removal of the Tris-EDTA buffer, rebanding of virus harvested at the heavy density resulted in its banding at the lower 1.16 g/cm³ density. The data indicate that density change could not be explained on the basis of the loss of some component from the virus or on the basis of the failure of the virus to reach equilibrium. However, it is possible that the two densities observed were a reflection of the existence of rubella virus in different hydration states in the presence and absence of Tris buffer containing EDTA.     

Purification and characterization of the hepatitis B virus core antigen produced in the yeast Saccharomyces cerevisiae

Hepatitis B virus core antigen gene was expressed in Saccharomyces cerevisiae and the product (yHBcAg) was purified from a crude lysate of the yeast by three steps: sucrose step-gradient ultracentrifugation, hydroxyapatite chromatography and CsCl-isopycnic ultracentrifugation. yHBcAg was synthesized in yeast cells as a particle consisting of polypeptides which have a molecular weight of 21.5 kDa (p21.5). In the CsCl-density gradient, yHBcAg particles synthesized with the expression vector pYG701c (the GAP promoter) had two peaks, at 1.35 g cm⁻³ (HP; high-density particle) and 1.31 g cm⁻³ (LP; low-density particle). On the other hand, the particles synthesized with expression vector pAC701 (the PHO5 promoter) had only one peak at 1.32 g cm⁻³. The isoelectric points of HP and LP were estimated to be 4.05 and 4.07, respectively. Absorption spectrum analysis showed that the HP contains nucleic acids. yHBcAg particles possessed the immunogenicity of HBcAg and its component polypeptide (p21.5) possessed that of HBeAg in addition to HBcAg. Moreover, Western blotting analysis of p21.5 using a monoclonal antibody against yHBcAg or yHBeAg indicated that the hepatitis B virus C-gene-coded protein shares the antigenic sites responsible for both antibodies.     

Purification and some properties of strawberry mottle virus

Strawberry mottle virus (SMoV) (three isolates: HJ, 3E and N) were transmitted to Chenopodium quinoa plants by sap inoculation. All three isolates induced very similar symptoms consisting of chlorotic spots and ringspots in inoculated leaves, and vein chlorosis, mottling, and dwarfing of the upper leaves. SMoV isolate HJ was purified from infected C. quinoa by homogenisation with 10 mM phosphate buffer, pH 7.2 containing 5% Triton X-100, followed by differential, sucrose density-gradient and CsCl equilibrium density-gradient centrifugations. A fraction with a buoyant density of 1.42g^- cm^-3 after CsCl density-gradient centrifugation was highly infectious to C. quinoa and contained many isometric virus-like particles c. 37 nm in diameter. These virus-like particles were never found in fractions from uninfected preparations. Electrophoretic analysis of a fraction containing virus-like particles revealed that these particles might have a single coat protein subunit with the apparent molecular mass of 26 K daltons and one nucleic acid of 6.6 kilobases. Double-stranded RNA analysis of isolate HJ-infected or uninfected C. quinoa and Fragaria vesca var. semperflorens seedling line ‘Alpine’ plants showed that both infected plants had two infection-specific dsRNA bands of mol. wts 4.5 and 3.9 × 10⁶.     

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.     

The gene transfer agent of Rhodopseudomonas capsulata,: Purification and characterization of its nucleic acid

Photosynthetic bacteria of the species Rhodopseudomonas capsulata are capable of exchanging genetic information via a recently discovered gene transfer agent (GTA). The 70S particle mediating the genetic exchange was purified and its nucleic acid was analyzed. Cell-free filtrates containing GTA were prepared by filtration of stationary cultures of R. capsulata on an Amicon thin-channel filtration apparatus. Purification of this filtrate was achieved by successive membrane filtration, diafiltration, agarose-gel filtration, ion-exchange chromatography on diethylaminoethyl cellulose, and sucrose gradient sedimentation, resulting in an overall purification of 4000-fold with a yield of 2–4%. [³H]thymidine-labeled nucleic acid isolated from this material was identified as deoxyribonucleic acid on the basis of its resistance to alkaline hydrolysis and its buoyant density of 1.718 g/ml in CsCl. The double-stranded nature of the deoxyribonucleic was demonstrated by its resistance to degradation by the single-strand-specific S₁-nuclease and the density shift in CsCl of +0.016 g/ml upon denaturation. Its molecular weight was estimated to be 3.6 × 10⁶ from sucrose gradient sedimentation in the presence of markers, and the linear, unnicked nature of the molecule was evident from sedimentation in an alkaline sucrose gradient.     

Identification and separation of components of calf thymus DNA using a CsCl-netropsin density gradient

Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated in calf thymus DNA. Satellite DNA's were isolated using preparative CsCl - netropsin gradient centrifugation and were characterised by density and homogeneity in native and in reassociated state. Two of the satellite components, with densities of 1.722 and 1.714 g/cm³, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm³ appear to be heterogeneous.