Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent

The scrapie agent causes a progressive degeneration of the central nervous system of animals after a prolonged incubation period. Measurements of incubation period length, defined as the time from inoculation to the onset of clinical signs of neurological dysfunction, were related to the titer of the agent and the dilution of the inoculated sample. Equations defining the relationship provide a new assay for the agent requiring fewer animals than end point titrations. By use of this incubation period assay, the scrapie agent from hamster brain was found to have an s20,w of < 300 S but > 30 S assuming rho p = 1.2 g/cm3. A partially purified fraction P3 was obtained by differential centrifugation and sodium deoxycholate extraction. When P3 was extracted with phenol, virtually no infectivity was found in the aqueous phase even after examining such variables as pH, salt concentration, and predigestion of samples with proteinase K. Nonionic and nondenaturing, anionic detergents did not inactivate the scrapie agent; in contrast, denaturing detergents inactivated the agent. Sodium dodecyl sulfate (NaDodSO4) inactivated greater than 90% of the agent at a NaDodSO4 to protein ratio of 1.8 g/g. Inactivation by NaDodSO4 appears to be a cooperative process. Addition of a nonionic detergent to form mixed micelles with NaDodSO4 prevented inactivation of the agent by NaDodSO4. Weak chaotropic ions do not inactivate the scrapie agent while strong chaotropic ions like SCN- and Cl3CCOO- destroy infectivity at concentrations of 0.2 M. These data provide evidence in support of a protein component within the scrapie agent which is essential for maintenance of infectivity. Thus, it is unlikely that the scrapie agent is composed only of a "naked" nucleic acid as is the case for the plant viroids.     

Disruption of prion rods generates 10-nm spherical particles having high alpha-helical content and lacking scrapie infectivity.

An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.     

Purification of scrapie agent from infected animal brains and raising of antibodies to the purified fraction

The fraction (P4) containing scrapie infectivity was obtained by treatment of scrapie-infected mouse brains with the detergent sarcosyl, differential centrifugation, and proteolytic enzyme digestion. Scrapie infectivity in the P4 fraction was purified 239-2,390 times with respect to protein. Similar fractions were also prepared from the brain of a sheep naturally infected with scrapie. Morphological observation of the P4 fractions revealed that the main components were unique rods of 3-5 X 60-200 nm, which resembled scrapie-associated fibrils (SAF) or prion rods. The P4 fractions formed three major broad bands of polypeptides with molecular weights (MWs) of about 24.5K, 21K, and 17K dalton (Kd) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some low MW polypeptides were also present in the fraction. Rabbits immunized with this fraction prepared from mouse brains raised antibodies against the three major polypeptides.     

Towards purification of the scrapie agent

A method for the partial purification of scrapie infectivity from hamster brain is described. About a 100-1000-fold, 20-fold, and 200-fold enrichment in scrapie infectivity with respect to protein, RNA, and DNA content has been achieved using differential centrifugation, enzyme and detergent treatment. The inbred CLAC strain of hamsters used in our experiments contained about 10 times less infectivity in brain than has been found in randomly bred animals or other inbred strains.     

Sedimentation characteristics of the scrapie agent from murine spleen and brain.

Sedimentation profiles of the scrapie agent in extracts of murine spleen and brain were determined by analytical differential centrifugation. Infectivity profiles of the agent from the two tissues were similar. Sedimentation of the agent was not substantially altered by detergent treatment with sodium deoxycholate. In the presence of detergent, centrifugation at an omega2t value of 3.0 x 1010 rad2/s in a fixed-angle rotor sedimented 90% of the agent. Comparative studies with radioisotopically labeled Simian virus 40 showed that centrifugation at an omega2t value of 1.6 x 10(10) rad2/s removed 90% of the virions. The sedimentation profile of the scrapie agent was similar to that observed for cellular ribosomal RNA. Heating infectious extracts of spleen to 80 degrees C for 30 min resulted in the destruction of 95% of the RNA while sedimentation of the scrapie agent was unchanged. These studies establish a limited range of particle sizes for the scrapie agent.     

Evidence of mitochondrial involvement in scrapie infection.

Two cDNA libraries were constructed from brain membrane and cytoskeletal preparations purified from scrapie-infected hamster brains. Four recombinants strongly preferential to the scrapie cytoskeletal preparation were identified by the differential hybridization of 7,000 recombinants. These clones were not, however, preferential to total nucleic acids extracted from scrapie-infected hamster brains. DNA sequence analysis revealed all four clones to have significant sequence similarities to the mouse mitochondrial genome. This correlation led us to consider a mitochondrial association with scrapie infectivity. Brain mitochondria were purified by sucrose gradient density centrifugation and found to contain high infectivity. Removal of mitochondrial outer membranes by osmotic shock or digitonin treatment resulted in no detectable loss of titer.     

Citrus psorosis is probably caused by a bipartate ssRNA virus

Isolate 90-1-1 Concordia (Argentina) of the citrus psorosis agent was graft-transmitted to citrus and mechanically transmitted to Chenopodium quinoa, which was used as a local lesion assay host. Infected citrus and C. quinoa plant lesions were used as starting materials for the purification of the psorosis-associated agent.In extracts partially purified by differential centrifugation, infectivity was abolished by RNase treatment, even in 0.3 M NaCl, indicating that ssRNA is required for biological activity. The total loss of infectivity produced by proteinase K treatment and the decline in infectivity caused by phenol extraction indicated that protein may be essential for infectivity.When partially purified extracts were subjected to sucrose density gradient centrifugation, infectivity on C. quinoa from certain 2-fraction combinations was higher than expected, compared to the infectivity of the individual fractions. Therefore, infectivity was not associated with a single component but with the combination of at least two components which were distinguishable on sedimentation.The infectious material was present in the top and bottom zones of a sucrose gradient, which, on further purification by a second gradient and agarose gel electrophoresis, revealed the presence of a 50-kDa protein. This protein was absent in comparable gradient fractions from healthy plants, and therefore most likely represented the capsid protein of both the top and bottom sucrose gradient zone components.Taken together, these results led to the conclusion that the citrus-psorosis-associated virus (CPsAV) is a multipartite virus, containing ssRNA and a 50-kDa coat protein. In view of the information available to date, CPsAV seems to be very closely related to citrus ringspot virus described in Florida (USA) and the psoriasis agent in Spain.     

Electron microscopic demonstration and isolation of ribosomes in mesosomes from Staphylococcus aureus

Mesosomes of Staphylococcus aureus 209P were observed to be extruded as tubules upon protoplast formation by electron microscopy and isolated under hypertonic conditions to maintain their structural integrity by differential centrifugation followed by sucrose density gradient centrifugation. Isolated mesosomes were composed of long, branched tubules of irregular sizes and they were shortened during purification. Thin sections of isolated mesosomes showed that the mesosomal tubule was surrounded by a triple-layered membrane and contained ribosome-like particles in diameter of about 15 to 20 nm. These particles were isolated from purified mesosomal preparation by disrupting the mesosomal tubule with deoxycholate and Triton X-100 under hypotonic conditions followed by a linear sucrose density gradient centrifugation. Negatively stained preparations of the isolated particles revealed the same appearance as those of the ribosomes isolated from the cytoplasm. The mesosomal particles sedimented at 70S in sucrose gradients in the presence of 10 mM Mg2+, but they were dissociated into two subparticles, 50S and 30S subunits, upon lowering the Mg2+ concentration to 1 mM. These findings indicate that the mesosomal tubule is packed with ribosomes.     

The isolation of endoplasmic reticulum from barley aleurone layers

Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13–1.14 g cc^-1 and 1.17–1.18 g cc^-1. Electron microscopy shows that the membranes sedimenting at 1.13–1.14 g cc^-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17–1.18 g cc^-1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - Trizma tris(hydroxymethyl)aminomethane     

ATTACHMENT OF RIBOSOMES TO MEMBRANES DURING POLYSOME FORMATION IN MOUSE SARCOMA 180 CELLS

Addition of nutrients to starved mouse S-180 cells leads to rapid conversion of ribosomal monomers to polysomes. During this process, a portion of the ribosomes originally found in the 17,000 g (10 min centrifugation) supernatant of cell lysates becomes firmly attached to structures sedimenting at 500 g (5 min centrifugation). Electron microscopy of sections of the intact cells showed the change from randomly distributed ribosomal particles to clusters. Association with membranes also became evident. The material sedimenting at 500 g comprised nuclei enclosed in an extensive endoplasmic reticulum (ER) network. This fraction prepared from recovering cells showed numerous ribosome clusters associated with the ER network. The appearance of many of these clusters indicated that the ribosomal particles were not directly bound to the membranes. RNase treatment released about 40% of the attached ribosomes as monomers, and ethylenediaminetetraacetic acid released 60% as subunits. It is suggested that during polysome formation a portion of the ribosomes becomes attached to the membranes through the intermediary of messenger RNA.     

Electron microscopic examination of ribosome preparations from germinated spores of Aspergillus fumigatus

Ribosomes were isolated from germinated spores (germlings) of Aspergillus fumigatus and electron microscopy was used to determine qualitatively the extent of cellular contamination. After differential centrifugation, the initial crude preparation contained 80S ribosomes and numerous membrane contaminants in the form of membrane sheets, palisade aggregates, and vesicles 0.10–0.18 m in size. Gel filtration chromatography of crude ribosomes in Sepharose CL-4B did not remove all of the membrane contamination. Homogenous ribosome suspensions, morphologically free of other cellular component contamination, were then obtained after gel filtration fractions were centrifuged through a 35% sucrose solution. The total ribosome yield from a germling preparation was approximately four times the yield of ribosomes from a comparable spore preparation. Gel diffusion precipitin patterns of germling ribosomes were identical to those of spore ribosomes with both germling and spore ribosomes antisera.     

Cellular content of ribonucleic acid and protein in Saccharomyces cerevisiae as a function of exponential growth rate: calculation of the apparent peptide chain elongation rate.

The average cellular content of ribonucleic acid and protein was determined in cultures of Saccharomyces cerevisiae growing exponentially at different rates in a variety of media. Estimations of the proportion of total cellular ribonucleic acid that is made up of ribosomal ribonucleic acid were used to calculate the average number of ribosomes per cell at the different growth rates. The fraction of ribosomes actively engaged in translation was estimated by sucrose gradient centrifugation of ribosomes and polysomes. These data were used in a calculation of the apparent time taken for the addition of an amino acid to the growing polypeptide chain; this value was found to vary linearly with growth rate over a fivefold range of doubling times.     

区带超速离心提纯流感病毒的初步探讨

在制备灭活流感疫苗中,采用两次蔗糖速率区带超离心从流感病毒感染的鸡胚尿液中提纯流感病毒是较简便、实用的方法,根据流感病毒分子特性设计了超离梯度并采用适当试验条件,获得了大量提纯流感病毒的初步结果。经血凝、电镜、蔗糖浓度、浮力密度等检验证明此方法是可行的。     

Protease sensitivity and nuclease resistance of the scrapie agent propagated in vitro in neuroblastoma cells.

The scrapie agent has been propagated in vitro in mouse neuroblastoma cells. To further characterize the tissue culture-derived scrapie agent, we studied the effects of protease and nuclease digestion on the agent derived from these cells. The scrapie agent in these cells was found to be resistant to protease digestions for short times but was inactivated by prolonged digestion at high protease concentrations. In contrast, digestion with a variety of nucleases did not alter the agent titer. These results demonstrate that the agent requires an essential protein or proteins for infectivity. If the agent also contains a nucleic acid genome, it must be more nuclease resistant than the majority of cellular DNA and RNA. These properties of the tissue culture-derived scrapie agent were identical to those of brain-derived scrapie agent and thus cannot be attributed to secondary effects of tissue pathology, since the infected cell cultures show no cytopathic effects as a result of infection.     

Stored mRNA in sporangiospores of the fungus Mucor racemosus.

When introduced into nutrient medium under air, the asexual sporangiospores of Mucor racemosus germinated with 5 to 8 h, culminating with the emergence of germ tubes. We found that sporangiospores increased 20% in dry weight during the first 60 min of germination, indicating a high degree of synthetic activity. Sucrose density gradient analysis of spore extracts revealed that the percentage of ribosomes associated with mRNA increased from 22.5% in dormant spores to 85% within 10 min after the addition of medium and remained at this level for at least 3 h. L-[14C]leucine was immediately incorporated at a rapid rate into protein of a leucine auxotroph, whereas [3H]uracil or [32P]phosphate was incorporated into RNA at a significant rate only 20 min after the addition of medium. This newly synthesized RNA occurred in polysomes only after 30 min had passed. Pool synthesized RNA occurred in polysomes only after 30 min had passed. Pool equilibration of the radioactive precursors was not limiting to these measurements. Polyadenylated RNA was isolated from dormant spores by oligodeoxythymidylic acid-cellulose chromatography and was found to comprise 3.3% of the total cellular RNA. Sucrose density gradient centrifugation revealed the polyadenylated RNA to be heterodisperse in size, ranging from 6S to 20S. It was concluded that M. racemosus sporangiospores contain preformed mRNA which is translated commencing immediately upon the addition of nutrient medium.     

Isolation of ribosome particles from meningopneumonitis organisms

In ribonucleic acid (RNA) extracted by phenol and sodium dodecyl sulfate from purified reticulate bodies of meningopneumonitis (MP) organisms, 21S, 16S, and 4S RNA were found by sucrose density gradient sedimentation analysis. When purified reticulate bodies were homogenized by sonic treatment or by treatment with sodium deoxycholate and were fractionated by differential centrifugation, more than 50% of the RNA was recovered in the fraction which was sedimented by centrifugation at 105,000 x g for 2 hr, but not at 13,000 x g for 20 min. From homogenates prepared in this manner, 50S and 30S particles containing RNA were isolated by sucrose density gradient centrifugation. These 50S and 30S particles were also found in lysates of cytoplasmic fractions of infected cells which were labeled by (32)P during 17 to 17.5 hr or 15 to 18 hr after infection. The synthesis of 50S and 30S particles was not inhibited by actinomycin D. When infected cells were homogenized in the presence of 0.01 or 0.02 m MgCl(2), 70S particles were isolated instead of 50S and 30S particles. When dialyzed against low concentrations of MgCl(2), the 70S particles dissociated to 50S and 30S particles. The base ratio of the 70S particles is very similar to that of 16S plus 21S RNA. The characteristics of the 70S, 50S, and 30S particles suggest that these are ribosome particles, similar to bacterial ribosomes.     

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl₂. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [¹⁴C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.     

DNA synthesis in circulating erythroblasts of anemic duck. Isolation and properties of nuclear and cytoplasmic-nonmitochondrial DNA.

In the circulating blood of anemic ducks, 5% of all erythroid cells synthesize DNA. Immature erythroblasts, at all stages of differentiation, synthesize DNA although to a varying degree, while reticulocytes and erythrocytes do not. In the erythroid cell population labeled in vitro 2 h with 32Pi, half of the labeled DNA sediments as small-molecular-weight molecules, suggesting that these molecules fail to integrate into the high-molecular-weight components. Labeled DNA is found in the cytoplasmic postmitochondrial fractions and it is in a form of deoxyribonucleoproteins which cosediment with ribosomes as well as subribosomal particles in sucrose gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosediment with ribosomes as well as subribosomal particles in sucorse gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosdeiment with ribosomal subunits in such a way that the larger the particle, the larger the molecular weight of the DNA cosedimenting with it. The specific radioactivity of the cytoplasmic ribosome-derived and postribosomal-particle-derived DNAs and the small molecular-weight nuclear DNA is similar and 10-20-fold higher than that of the bulk nuclear DNA. The former three DNA species sediment between 4-14 S. It is concluded that the cytoplasmic nonmitochondrial DNA species are of the nuclear origin. Less than 0.5% of the total cellular nonmitochondrial DNA can be purified from the nucleus and the cytoplasm as fast-labeled small-molecular-weight components. All of the cellular nonmitochondrial DNA species band at the same mean buoyand density in Cs2SO4/urea gradients. All behave as native structures in hydroxyapatite and contain less than 5% of their length as single-stranded regions.     

THE PREPARATION AND PROPERTIES OF BACTERIAL CHROMATOPHORE FRACTIONS

Chromatophore material from the bacterium Rhodopseudomonas spheroides was freed of ribosomes by centrifugation in 27 per cent RbCl and then separated into "heavy" and "light" fractions by centrifugation through a sucrose gradient. The fractions differed from one another in the following ways. (a) The isopycnic density of the heavy fraction was between 1.15 and 1.18 gm/ml and that of the light fraction was 1.14 gm/ml. (b) The heavy fraction was able to bind ribosomes; the light fraction was not. (c) The light fraction was homogeneous in the ultracentrifuge and had a sedimentation constant, extrapolated to infinite dilution, of 153 s_20,w. The heavy fraction was grossly heterogeneous. (d) Both the amount of bacteriochlorophyll relative to protein and the ratio of bacteriochlorophyll to carotenoids were greater in the light fraction. (e) The spectra of the two fractions in the near infra-red were different. Comparisons of the chromatophore fractions from cells with different amounts of bacteriochlorophyll showed that the specific bacteriochlorophyll contents of the two fractions did not change to the same extent as did that of the whole cells. The amount of heavy fraction from pigmented cells was roughly independent of the cellular pigment content and was about equal to that from pigment-free cells. The amount of light fraction depended on the pigment content of the cells; no light fraction was obtained from cells devoid of bacteriochlorophyll. The cytochrome complements of both fractions underwent quantitative as well as qualitative changes with varying growth conditions. The size of the photosynthetic unit in R. spheroides appeared to increase as the total cellular bacteriochlorophyll content increased; however, the number of units per light fraction particle remained constant.