Characterization of the Snow Mountain agent of viral gastroenteritis.

Snow Mountain agent (SMA) is a 27- to 32-nm virus which is the etiologic agent of outbreaks of acute gastroenteritis in Colorado and Vermont. SMA is morphologically similar to but antigenically distinct from the Norwalk and Hawaii agents of viral gastroenteritis but, like those agents, has not been cultivated in vitro. We purified and characterized SMA directly from human stool specimens containing the virus. The density of the SMA virion was 1.29 g/cm3 and 1.21 to 1.22 g/cm3 on potassium tartrate-glycerol gradients and 1.33 to 1.34 g/cm3 on cesium chloride gradients. SMA had an S value of 170 to 183S on a sucrose velocity gradient. The purified virion was iodinated, immunoprecipitated with acute and convalescent sera from volunteers challenged with SMA, and analyzed on polyacrylamide gels. The virion contains one major structural protein of 62,000 molecular weight, which is similar in size to the 59,000-molecular-weight protein found in the Norwalk virion. The biophysical properties and single structural protein of SMA most closely resemble those of the calicivirus group.     

Isolation of virus-like particles of in vitro cultures of Plasmodium falciparum

We have isolated virus-like particles from in vitro cultures of the malarial agent Plasmodium falciparum. These particles have a buoyant density of 1.16 g/cm3, contain DNA, and appear to arise from budding structures on the surface of parasitized erythrocytes.     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

罗氏沼虾体内两种病毒颗粒的分离、纯化与核酸特性

从患肌肉白浊症状的罗氏沼虾幼苗体内分离纯化得到两种大小不同的病毒颗粒.这两种病毒颗粒均为对称的20面体结构,表面光滑,无囊膜,对氯仿不敏感.一种是直径为26nm～27nm的颗粒,在氯化铯中的密度为132g/cm3,病毒基因组含两段单链的RNA,分别为30kb和12kb,具有诺达病毒科成员的特征.一种是直径为14nm～16nm的颗粒,在氯化铯中的密度为133g/cm3,含有一段大小为09kb的单链RNA,拟为卫星病毒样颗粒或辅助病毒.     

Dynamics of the accumulation of virus particles with different physico-chemical properties in FRhK-4 cells infected with hepatitis A virus

The propagation time-course of hepatitis A virus (HAV, strain HAS-15) in continuous culture of the foetal rhesus monkey kidney cells (FRhK-4) was investigated. The HAV infectivity and viral RNA content in the infected cells reached the maximal level 5-8 days after infection, while accumulation of hepatitis A antigen (HAAg) continued for 2-3 weeks more. Viral particles with the densities 1.27-1.28 g/cm3 and 1.18-1.22 g/cm3 were isolated from the infected cells as well as the mature virions with the buoyant density 1.33-1.34 g/cm3 in CsCl. The concurrent accumulation of mature virus and "light" particles (1.18-1.22 g/cm3) was registered during infection. Viral particles with the density 1.27-1.28 g/cm3 accumulated predominantly from the 14th to the 21st-24th days after infection. The mature virions (1.34 g/cm3) as well as the particles with the density 1.24-1.25 g/cm3 were isolated from supernatant precipitated by ammonium sulphate. The HAAg activity of both fractions increased progressively in equal proportion in course of infection.     

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.     

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.     

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.     

Effects of tumor promoters on the rate and commitment to terminal differentiation of subpopulations of murine keratinocytes

The effects of skin-tumor-promoting and -nonpromoting agents on the kinetics of terminal differentiation of subpopulations of keratinocytes differing in buoyant density isolated from mice (SENCAR) that are very sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion were investigated. Topical pretreatment of dorsal skin with complete (TPA), first-stage (calcium ionophore A23187) and second-stage (mezerein) tumor promoters, but not the hyperplastic agent ethylphenylpropiolate, accelerated the rate of terminal differentiation of keratinocytes with densities less than 1.074 g/cm3, but had little effect on cells with a greater density. Within 8.5 hr of TPA treatment, a period preceding mitosis, a large percentage of the most dense basal-cell keratinocytes (greater than or equal to 1.074 g/cm3) were converted to cells with a lower density, with a reduced plating efficiency and with an increased rate of differentiation, suggesting that TPA induces a subpopulation of basal cells to commit to terminal differentiation, and accelerates the rate of differentiation of committed cells.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.

Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.     

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.     

Specific Labeling and Physical Characterization of R-Factor Deoxyribonucleic Acid in Escherichia coli

The molecular nature of R-factor deoxyribonucleic acid (DNA) was examined in Escherichia coli by using a method for the specific labeling of the derepressed R factor, R1, in a female cell after conjugation. Sixty minutes after mating, the R factor was isolated as a single molecule with a molecular weight of 65 x 10(6) daltons. This single molecular species sedimented as either a covalently closed molecule or a "nicked" circle. When the single R-factor component was centrifuged in a CsCl density gradient, only a single homogeneous species with a buoyant density of 1.711 g/cm(3) was observed. R-factor DNA was also isolated directly from exponentially growing cells of E. coli as a covalently closed single molecular species comprising about 1% of the total cellular DNA. Previous studies in Proteus show that R1 factor DNA components of buoyant density 1.709, 1.711, and 1.716 g/cm(3) can be identified as distinct replicons. It is suggested that the single molecule of R1 observed in E. coli is most simply explained as a composite structure resulting from a recombinational assemblage of a 1.709 and 1.716 g/cm(3) replicon.     

On the question of integration of Agrobacterium tumefaciens deoxyribonucleic acid by tomato plants.

Treatment of tomato plants with Agrobacterium tumefaciens causes subsequently administered [3H]thymidine to be preferentially incorporated into a satellite deoxyribonucleic acid (DNA) whose buoyant density is between that of bacterial DNA (rho = 1.718 g/cm3) and plant main band DNA (rho = 1.692 g/cm3). Satellite DNA upon shearing or sonic treatment releases fragments of higher and lower buoyant density, as reported by earlier investigators. The satellite has no significant base sequence homology with A. tumefaciens DNA, for its rate of reassociation is not accelerated by the addition of high concentrations of the latter. Tomato DNA isolated from shoots or from leaf nuclei accelerates renaturation of labeled satellite DNA. We conclude that the intermediate density labeled DNA is a plant satellite and not the product of covalent joining of bacterial and plant DNA as suggested by earlier investigators.     

Physical characterization of Rhizobium meliloti megaplasmids

Intact megaplasmids of Rhizobium meliloti 2011 have been isolated and visualized by electron microscopy. The contour lengths of 64 megaplasmid molecules were determined. One definite class of molecules of 400 micron length and a range of larger molecules with lengths of up to 560 micron was observed. The contour lengths of the megaplasmids pRme2011a and pRme2011b were measured after isolation from plasmid-free Agrobacterium strains into which they had been individually transferred. Plasmid pRme2011a corresponds to the 400-micron class of megaplasmids while plasmid pRme2011b belongs to the 560-micron class. Preparatively isolated megaplasmids pRme2011a and b showed completely different restriction patterns. The pattern of total megaplasmid DNA from R. meliloti 2011 is composed of those from pRme2011a and b, suggesting that no more than two different megaplasmids exist. Because the length distributions of measured molecules were broad, R. meliloti 2011 megaplasmids seem to vary in length in vivo. Because only pRme2011a hybridized with a nifHD probe, this is the Sym plasmid. For R. meliloti strain MVII-1, which carries the megaplasmids pRmeMVII-1f and pRmeMVII-1g, pRmeMVII-1f was shown to be the Sym plasmid. Buoyant density determinations of R. meliloti 2011 and MVII-1 megaplasmids gave a value of 1.717 g/cm3 for pSym, which is that of Agrobacterium DNA. The buoyant density of the second megaplasmid was 1.721 g/cm3, corresponding to the density of the R. meliloti chromosome. As determined by reassociation kinetics, pRme2011a and b are unrelated. The degree of relatedness between strains MVII-1 and 2011 was 82%.     

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.     

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.     

Properties of satellite DNA of three plant species of the subtribe Citrinae

The properties of stDNA of three species of the subtribe Citrinae have been investigated. The buoyant density of the main component is 1.693 g/cm3, that of satellite component is 1.712 g/cm3 and 1.715 g/cm3. The differential melting curves of satellite components reveal two melting zones. Some of stDNAs are melted within a broad temperature range, while others--at higher temperatures as a narrow peak. The reassociation kinetics suggest that 50-54% of stDNA are a fast reassociating fraction with the length of repeating sequences of 0.8-1.1 x 10(2) base pairs. Based on the values of Tm and buoyant density the 5-methylcytosine content in stDNA was calculated and was found equal to 20-35%. Using equilibrium ultracentrifugation in the actinomycin D--CsCl density gradient the stDNAs of the subtribe Citrinae were separated into constituent components.     

Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane

We have used a combined biochemical and morphological approach to establish the suitability of certain endogenous and exogenous domain markers for monitoring the separation of rat hepatocyte plasma membrane domains in sucrose density gradients. As endogenous domain markers, we employed two of the integral plasma membrane protein antigens, HA 4 and CE 9, localized to the bile canalicular and sinusoidal/lateral domains, respectively, of the hepatocyte plasma membrane in rat liver tissue (Hubbard, A. L., J. R. Bartles, and L. T. Braiterman, 1985, J. Cell Biol., 100:1115-1125). We used immunoelectron microscopy with a colloidal gold probe to demonstrate that HA 4 and CE 9 retained their domain-specific localizations on isolated hepatocyte plasma membrane sheets. When the plasma membrane sheets were vesiculated by sonication and the resulting vesicles were centrifuged to equilibrium in sucrose density gradients, quantitative immunoblotting revealed that the vesicles containing HA 4 and those containing CE 9 exhibited distinct density profiles. The density profile for the bile canalicular vesicles (marked by HA 4) was characterized by a single peak at a density of 1.10 g/cm3. The density profile for the sinusoidal/lateral vesicles (marked by CE 9) was bimodal, with a peak in the body of the gradient at a density of 1.14 g/cm3 and a smaller amount in the pellet (density greater than or equal to 1.17 g/cm3). We used this sucrose gradient fractionation as a diagnostic procedure to assign domain localizations for several other hepatocyte plasma membrane antigens and enzyme activities. In addition, we used the technique to demonstrate that 125I-wheat germ agglutinin, introduced during isolated liver perfusion at 4 degrees C, can serve as an exogenous domain marker for the sinusoidal domain of the rat hepatocyte plasma membrane.     

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.