Altered Sedimentation Research Articles

Altered Collection Interfaces in Hematopoietic Progenitor Collection of Sickle Cell Patients

Abstract Sickle cell disease (SCD) results from a point mutation in the beta globin gene leading to abnormal hemoglobin production and, correspondingly, abnormal sickling of erythrocytes that result in chronic anemia and vaso-occlusive crises. Patients with sickle cell are commonly treated with lifelong transfusions or hydroxyurea to increase fetal hemoglobin to ameliorate sickling. Evolving therapies are aimed at correcting known mutations with gene therapy on autologous hematopoietic stem and progenitor cells (HSCs), indicating a growing need for optimal stem cell collections from SCD patients. Recent studies have shown the safety and efficacy of plerixafor to increase peripheral CD34+ cells, enhancing collection. Here we show that standard apheresis collection procedures from sickle cell patients are inefficient when compared to healthy donors. Eleven patients with SCD were recruited to receive plerixafor and followed by peripheral CD34+ cell monitoring and apheresis collection. Overall, efficiency ranged from 2% to 55% with no correlation to total peripheral CD34+ cell count. To better understand where the CD34+ cells sedimented in the apheresis instrument, we collected different layers of the interface ranging from 3% to 10% estimated Hct and found that deeper layers with higher hematocrit (7.5%-10%) are enriched for CD34+ cells when compared to historical donors with healthy red cells. All patients had undergone red cell exchange prior to collection, yet this intervention did not prevent the altered sedimentation of CD34+ cells. These findings indicate that CD34+ cells from SCD patients sediment at a deeper, higher Hct interface layer during apheresis and support the altered collection practices for the efficient collection of HSCs for cellular therapies.

Arabidopsis Myosins XI1, XI2, and XIK Are Crucial for Gravity-Induced Bending of Inflorescence Stems.

Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis.

Late Holocene high precipitation events recorded in lake sediments and catchment geomorphology, Lake Vuoksjávrátje, NW Sweden

In this paper, we show the potential of combining multi‐proxy analysis of lake sediments with catchment geomorphology to better understand palaeoenvironmental changes. Previous studies have suggested that alpine lakes in N Sweden may be highly sensitive to variations in catchment erosion and precipitation, and that this sensitivity may influence ecologically based reconstructions of past temperature changes. We analysed lake sediments covering the last 5100 years from the alpine Lake Vuoksjávrátje in NW Sweden in order to identify different erosional regimes in the lake catchment, sediment sources and lake sedimentary processes, which ultimately affect the palaeoecological record. The measured proxies include elemental geochemistry from XRF core scanning, grain size, sediment accumulation rates, fraction of terrestrial organic carbon and geomorphological mapping, supported by previously published chironomid and total organic carbon data. From the integrated results we identified time intervals when increased intensity of precipitation altered sedimentation and lake catchment erosional processes. The most prominent event occurred c. 2900 cal. a BP and is interpreted to be the result of excessive precipitation, possibly related to the climatic shift towards cooler and wetter conditions referred to as the 2.8 ka event.

Pool‐fills: a window to palaeoflood history and response in bedrock‐confined rivers

AbstractChannel‐scale sedimentary units associated with bedrock‐controlled riffle‐pool morphology are examined in detail along Sandy Creek gorge, an ephemeral stream in arid south‐eastern central Australia. Pool‐fills comprise cut‐and‐fill assemblages of poorly sorted sediments ranging in texture from muds to boulders. Five unit types are defined based on particle size, sedimentary structures, geometry and bounding surface character: (1) coarse‐grained bar platform; (2) fine‐grained bar supraplatform; (3) fine‐grained pool‐fill; (4) fine‐grained bench; and (5) modern pool‐fill. The last coarse‐grained unit currently lining the pools suggests an altered sedimentation style over the post‐settlement period (post‐ad 1860s). Situated at bedrock valley constrictions, pool‐fills are compared with other sedimentary units associated with recirculating currents: eddy bars and slackwater deposits. But only the fine‐grained bench units reflect eddy recirculation; the pool‐fills are principally forced‐bars associated with bedrock‐controlled or ‘forced’ riffle‐pool morphology. A late Holocene palaeoflood history is proposed based on radiocarbon ages from the pool‐fills: multiple phases of cut‐and‐fill activity were preceded by a superflood 3400–1900 years ago that eroded the pool‐fills to bedrock. The resilience of the pool‐fills was illustrated by the passage of a 1‐in‐100‐year flood in 1992, which caused only minor erosion. The presence of pool‐fills may provide a window to past phases of river activity that cannot be extracted from either historical records/observations or palaeoflood slackwater sediment analyses. The formation and sedimentary preservation potential of these landforms reflect a combination of hydraulic and structural influences, but the occurrence of high‐magnitude floods exerts the dominant control.

Echovirus infection of rhabdomyosarcoma cells is inhibited by antiserum to the complement control protein CD59.

A number of echoviruses use decay accelerating factor (DAF) as a cellular receptor or attachment protein for cell infection. Binding of echovirus 7 to DAF at the cell surface, but not to soluble DAF in solution, triggers the formation of virus particles exhibiting an altered sedimentation coefficient ('A' particles) which are considered indicative of the particle uncoating process. We have previously demonstrated that antibodies to beta(2)-microglobulin block cell infection at a stage prior to 'A' particle formation and suggested that this reflects the involvement of beta(2)-microglobulin (or the associated MHC-I) in a virus-receptor complex that forms at the cell surface. We demonstrate here that antiserum to CD59 specifically blocks infection of rhabdomyosarcoma cells by a range of echoviruses, including viruses that bind DAF (e. g. echovirus 7) and those that use currently unidentified receptors other than DAF. The block occurs prior to 'A' particle formation and is cell-type specific. The potential role of CD59 as an active member, or passive participant, in the virus-receptor complex is discussed.

A Ser162→ Leu Mutation within Glycoprotein (GP) IIIa (Integrin β3) Results in an Unstable αIIbβ3 Complex that Retains Partial Function in a Novel Form of Type II Glanzmann Thrombasthenia

Platelets from Glanzmann thrombasthenia patient BL express approximately 30% of the normal alphaIIbbeta3 content and support fibrin-mediated clot retraction, but fail to bind fibrinogen or aggregate following cellular activation. BL platelets bind neither activation-dependent nor activation-independent ligands. DNA sequence analysis of BL platelet mRNA revealed a homozygous C583-->T point mutation in a conserved region of beta3, resulting in a Ser162Leu amino acid substitution. This mutation appears to produce destabilizing effects on the alphaIIbbeta3 complex, as evidenced by the fact that (1) the BL alphaIIbbeta3 complex exhibited altered sedimentation velocity through sucrose gradients, (2) alphaIIb and beta3 was not recognized by complex-dependent monoclonal antibodies or co-precipitated by integrin subunit-specific antibodies, and (3) biosynthesis and trafficking of the alphaIIbbeta3Leu162 complex was delayed relative to that of the wild-type control. Taken together, these data implicate the region encompassing Ser162 in the stabilization and ligand binding properties of the alphaIIbbeta3 complex.

A search for evidence of virus/transgene interactions in potatoes transformed with the potato leafroll virus replicase and coat protein genes

A search was conducted to detect evidence for interactions between potato leafroll virus (PLRV)-derived transgenes expressed in Russet Burbank potato and viruses to which the transgenic plants were exposed and by which they were infected. More than 25000 plants in 442 lines transformed with 16 different coat protein gene (CP) constructs and nearly 40000 plants in 512 lines transformed with seven different replicase gene (Rep) constructs of PLRV were exposed to field infection over a 6-year period. These plants were individually inspected for type and severity of virus symptoms. Heterologous viruses found infecting the plants were identified and examined for alterations in transmission characteristics, serological affinity, host range, and symptoms. Selected isolates of PLRV from field-infected plants were examined for unusual symptoms produced in diagnostic hosts and for alteration in sedimentation properties in density gradient tubes. Viruses that were propagated in selected transgenic lines in a greenhouse were examined for similar alterations. Transmission characteristics and serological properties were not altered when they replicated in potatoes containing CP constructs in the field or greenhouse. Potato plants expressing CP or Rep constructs of PLRV were not infected in the field or in the greenhouse with viruses that do not normally infect potato. New viruses or viruses with altered sedimentation characteristics, symptoms, or host range were not detected in field-exposed or greenhouse-inoculated potato plants expressing CP or Rep gene constructs of PLRV.

Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits.

Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C-terminal part (mutants beta delta 209-215, beta delta 194-215, beta delta 181-215, beta delta 171-215, beta delta 150-215) or have undergone Ala substitutions for the acidic and basic residues which are concentrated in the sequences 55-70 and 171-180, respectively. All these mutants have been examined for their ability to functionally replace the wild type beta-subunit. All substitutions and the deletions delta 1-4, delta 194-215, and delta 209-215 are compatible with effective binding of the catalytic alpha-subunit, as judged by sucrose density gradient analysis, stimulation of catalytic activity, and protection against thermal denaturation. Deletions delta 171-215 and delta 150-215, however, give rise to truncated molecules which are unable to associate with the alpha-subunit. The intermediate deletion delta 181-215 is still compatible with association, albeit the reconstituted holoenzyme exhibits an altered sedimentation coefficient. The holoenzymes reconstituted with substituted mutants beta A 55,57, beta A55-57, and, to a lesser extent, beta A 59-61, beta A63,64, and beta A5,6 display a basal activity which is higher (up to 4-fold) than that of the wild type holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

The alpha subunit of the stimulatory guanine-nucleotide-binding regulatory protein forms high-molecular-mass aggregates, concomitant with iloprost-induced desensitization of human platelet adenylyl cyclase.

Prolonged treatment of human platelets with the prostacyclin analog iloprost led to desensitization of the response to various prostaglandin derivatives. However, basal adenylyl cyclase activity and stimulation by agents acting directly via Gs, the stimulatory guanine-nucleotide-binding regulatory protein of adenylyl cyclase, were likewise decreased. Reconstitution of desensitized membranes with purified Gs from turkey erythrocytes indicated no alteration in the catalyst itself. However, the function of Gs (in cholate extracts) appeared to be severely impaired when reconstituted with adenylyl cyclase catalyst. Modification of Gs was also indicated by its altered sedimentation in sucrose density gradients. From Western blots, the alpha subunit of Gs, alpha s, from control platelets sedimented as a 5.6S species, while that from desensitized cells appeared at higher S values (in a polydisperse distribution). Activation by guanosine 5'-[gamma-thio]triphosphate of Gs from control platelets shifted alpha s to 3.5-3.7S, while activation of Gs from desensitized platelets induced such shift only for a minor portion of alpha s. This small fraction alone appeared to be susceptible to ADP-ribosylation by cholera toxin/[32P]NAD. Furthermore, an antibody directed against the C-terminal hexadecapeptide of alpha s precipitated much less alpha s from cholate extracts derived from desensitized platelets. Modification of alpha s during desensitization was also suggested from cross-linking experiments using the homobifunctional agent bismaleimidohexane: alpha s from desensitized platelets formed a single product of 80 kDa, while that from untreated platelets yielded a doublet (100 kDa and 110 kDa).

Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation.

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.

Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds

The oligomeric structure of the influenza A virus M2 integral membrane protein was determined. On SDS-polyacrylamide gels under nonreducing conditions, the influenza A/Udorn/72 virus M2 forms disulfide-linked dimers (30 kDa) and tetramers (60 kDa). Sucrose gradient analysis and chemical cross-linking analysis indicated that the oligomeric form of M2 is a tetramer consisting of either a pair of disulfide-linked dimers or disulfide-linked tetramers. In addition, a small amount of a cross-linked species of 150–180,000 kDa, which the available data suggest contains only M2 polypeptides, was observed. The role of M2 cysteine residues in disulfide bond formation and their role in forming oligomers were examined by converting each of the two extracellular and single cytoplasmic cysteine residues to serine residues and expressing the altered M2 proteins in eukaryotic cells. Removal of either one of the N-terminal cysteines at residues 17 or 19 indicated that tetramers formed that consisted of a pair of noncovalently associated disulfide-linked dimers, suggesting that each of the cysteine residues is equally competent for forming disulfide bonds. When both cysteine residues were removed from the M2 N-terminal domain, no disulfide-linked forms were observed. When solubilized in detergent this double-cysteine mutant lost reactivity with a M2 specific mAb and exhibited an altered sedimentation pattern on sucrose gradients. However, chemical cross-linking of this double-cysteine mutant in membranes indicated that it can form tetramers. Taken together, these data suggest that disulfide bond formation, although not essential for oligomeric assembly, stabilizes the M2 tetramer from disruption by detergent solubilization.

Human progesterone receptor complexed with the antagonist RU 486 binds to hormone response elements in a structurally altered form.

Structural and functional properties of human progesterone receptors (PR) bound with the antiprogestin, RU 486, and the progestin agonist, R5020, were compared in order to identify receptor mechanisms responsible for the inability of RU 486 to activate the transcriptional capacity of receptors. RU 486 interaction with human PR did not inhibit receptor transformation as assessed by dissociation of nontransformed 8-10S oligomeric receptors (in vitro and in vivo) and by tight binding of PR to nuclei/chromatin in whole cells. Assays based on immunoprecipitation of PR-DNA complexes with an antibody to human PR and gel retardation were used to analyze the effect of RU 486 on receptor binding to the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). RU 486 did not impair PR recognition of the MMTV HRE. Quantitative affinity constants and kinetic parameters of PR binding to these specific DNA sites were similar for receptors complexed with either agonist or antagonist. However, PR-RU 486 complexes exhibited an altered sedimentation rate on sucrose gradients and a faster mobility when bound to the MMTV HRE as assessed by gel retardation. These results indicate that human PR transformed by RU 486 exhibit no impairment in binding to specific DNA sites of target genes, but when bound to DNA assumes a structural form different from that of the receptor-agonist complexes to activate transcription results from this structural alteration in PR, which does not permit protein-protein interactions required for receptor-mediated induction of gene transcription.

The assay, purification and properties of vaccinia virus-induced uncoating protein.

Vaccinia virus cores prepared in vitro can be uncoated by incubation with extracts of cells infected with vaccinia virus, as determined by the conversion of the genome to DNase susceptibility. This uncoating activity had all the characteristics of the corresponding in vivo activity and of the agent responsible for non-genetic reactivation. Thus, it was not induced by heat-inactivated virus, nor was it produced in the presence of inhibitors of RNA or protein synthesis. The uncoating protein induced by cowpox virus will uncoat vaccinia virus cores. The uncoating protein was concentrated from infected cell extracts by ultrafiltration and purified by gel filtration and ion-exchange and affinity chromatography. It was characterized as a trypsin-like protease with a mol. wt. of 23,050. Cores treated with the purified uncoating protein had an altered sedimentation rate but no differences between treated and untreated cores were detected by electron microscopy or polyacrylamide gel electrophoresis.

Retro-endocytosis of low density lipoprotein by cultured human skin fibroblasts.

A fraction of the low-density lipoprotein (LDL) internalized by cells via receptor-mediated endocytosis follows a short-circuit pathway, termed "retro-endocytosis," that results in the rapid exocytosis of ligand. Results from the current study suggest that retro-endocytosis of LDL in human fibroblasts is caused by resurfacing of endocytotic vesicles that contain both free and receptor-bound ligand, resulting in discharge of vesicular contents and in spontaneous dissociation of LDL from its receptor. The bulk of the released LDL particles had the same size, density, and immunogenic properties as native LDL, indicating that they were discharged intact. Some of the retro-endocytosed LDL was larger than native LDL, and some exhibited altered sedimentation properties. When fusion of endosomes with lysosomes was inhibited by chilling cells to 18 degrees C, the proportion of intracellular LDL subsequently released was unaffected, suggesting that retro-endocytosis does not require lysosomal participation. Furthermore, the shorter the internalization phase the greater was the proportion of LDL subsequently released, suggesting that LDL was discharged from compartments formed early in endocytosis. Retro-endocytosis of LDL was stimulated by agents that neutralize acid intracellular compartments, such as ionophores (monensin) and weak bases (chloroquine and methylamine). Monensin increased the proportion of intracellular LDL released, suggesting that it had a direct effect on retro-endocytosis. The effect of weak bases appeared to be secondary to their ability to promote cellular accumulation of undegraded LDL. Thus, retro-endocytosis of LDL becomes a major pathway when intracellular compartments fail to maintain a low pH or where the intracellular concentration of LDL reaches abnormal levels.

Sedimentation behavior of activated human granulocytes. Aggregation and volume effects.

Human peripheral blood granulocytes (PMNs) obtained from normal adults were studied by an analytical gravity sedimentation system. Exposure of PMNs to endotoxin-activated serum (EAS) in a Ficoll density gradient containing Hank's balanced salt solution with calcium and magnesium produced significantly different sedimentation patterns compared to those from granulocytes exposed to normal serum under the same conditions. Experiments were performed to determine whether changes in granulocyte density, volume, shape, or aggregation were responsible for the sedimentation pattern of granulocytes exposed to EAS. The altered gravity sedimentation behavior of endotoxin-activated granulocytes was abolished when calcium and magnesium were not present in the Ficoll density gradient. Granulocyte aggregation was inhibited by the absence of calcium and magnesium in the medium during granulocyte stimulation, whereas the changes in granulocyte shape and volume associated with granulocyte stimulation were not affected. The data indicate that the altered granulocyte sedimentation pattern in the presence of EAS and calcium and magnesium was produced by granulocyte aggregation and not by changes in granulocyte volume or shape.

Interaction of pyridoxal phosphate with glucocorticoid receptors from HeLa S3 cells.

HeLa S3 cells contain high affinity, saturable receptors which are specific for glucocorticoids. Cytoplasmic dexamethasone receptor complexes from these cells sedimented as a single peak, approximately 8.0 S in 5, to 20% sucrose gradients prepared in 10 mM Tris-HCl buffer, pH 7.4. Treatment of the dexamethasone receptor complexes with 5 mM pyridoxal phosphate caused a reduction in the size of the 8.0 S peak and the appearance of a second peak sedimenting at 5.7 S. Addition of NaBH4 following pyridoxal phosphate treatment resulted in a single new radioactive peak of the steroid receptor complex which sedimented at 3.5 S. Recentrifugation of the 3.5 S peak did not result in an altered sedimentation profile. However, recentrifugation of either the 8.0 S peak from untreated cytosol or the 5.7 S peak from pyridoxal phosphate treated cytosol resulted in a single peak which sedimented at 8.0 S. These results suggest that 5 mM pyridoxal phosphate causes the reversible conversion of the dexamethasone receptor complex to a smaller receptor form. This conversion is made irreversible by the addition of NaBH4, resulting in a new form of dexamethasone receptor complex sedimenting at 3.5 S. This alteration in receptor sedimentation coefficient was dependent on the time of incubation with pyridoxal phosphate prior to the reduction with NaBH4. In the presence of 5 mM pyridoxal phosphate, the decrease in [3H]-dexamethasone associated with 8.0 S dexamethasone receptor complexes followed a single exponential function with a rate constant of 1.63 × 102 min1. The shift in glucocorticoid receptor sedimentation coefficient was also dependent on the concentration of pyridoxal phosphate with which the receptor was incubated and was apparent at pyridoxal phosphate concentrations as low as 1 mM. In addition, it was highly specific for the physiologically active form of coenzyme. Treatment of cytoplasmic dexamethasone receptor complex with 5 mM pyridoxine, pyridoxal, pyridoxamine or pyridoxamine 5'-phosphate did not result in an alteration in receptor sedimentation coefficient. Unlike the 8.0 S dexamethasone receptor complex observed in untreated cytosols, this small form of receptor produced by treatment with pyridoxal phosphate and NaBH4 sedimented at 3.5 S independently of pH and ionic strength. The 3.5 S receptor may be a subunit or nonaggregated form of receptor derived from the larger 8.0 S complex. This small 3.5 S form of receptor has a calculated molecular weight of 42,000 daltons.

Further characterization of a population of defective, interfering pseudorabies virions

Defective, interfering particles of pseudorabies virus isolated after repeated (49) passages in rabbit kidney cells have been further characterized. The defective particles sediment at a slower rate in sucrose gradients than do standard virions. This difference in sedimentation rates is not due to a difference in the molecular weights of the DNA present in defective and standard virions; both types of particles contain DNA with a molecular weight of approximately 90 × 10 6. The content of DNA relative to protein is also the same for both types of particles (10–11 μg of DNA/100 μg of protein). Populations of purified defective and standard virions are homogeneous with respect to their content of DNA and protein. The defective particles become more highly labeled with glucosamine than do standard particles and the degree of glycosylation of some of the glycoproteins of defective particles differs from that of standard particles. The changed sedimentation behavior of defective virion populations may reside in the greater degree of glycosylation of some of their glycoproteins. It has proved impossible to separate by sedimentation the infectious virus present in the population of defective virions from the defective virions. The altered sedimentation behavior seems to be acquired by both infectious and defective particles produced by cells coinfected with defective and infectious virus.

Antiviral action of camptothecin.

At a concentration of 10 mum, camptothecin inhibited vaccinia deoxyribonucleic acid (DNA) synthesis in HeLa cells. Inhibition of viral DNA synthesis was observed when the drug was added before infection or at 1 or 2 hr after infection. Inhibitory effects of camptothecin on vaccinia DNA synthesis could be reversed, even after exposure to the alkaloid for 2 hr. Viral DNA, isolated from vaccinia-infected, camptothecin-treated cells, displayed an altered sedimentation constant after alkaline sucrose density gradient centrifugation. Incorporation of uridine into vaccinia messenger ribonucleic acid was inhibited by camptothecin, but the activity of ribonucleic acid polymerase, as tested in isolated vaccinia cores, was not affected by the drug. Camptothecin had essentially no effect on replication of poliovirus in HeLa cells.

Specific dissociation of pea 25-S rRNA by hot-phenol treatment

Purified pea 25-S rRNA was converted rapidly to the 17–18-S product by heating with phenol at 60°. This altered sedimentation behavior was not reversed by centrifuging the thermal product in sucrose gradients made up in high ionic strength buffers. Similar conversion of the 25-S to the 17–18-S product was brought about by exposure to 80 % dimethyl sulfoxide at o°. Nucleolar 26-S RNA, one of the ribosomal precursor molecules, was shown to be rather resistant to the specific degradation by hot-phenol treatment, as compared with cytoplasmic 25-S rRNA. Newly formed 25-S rRNA obtained from cytoplasm, as well as the old 25-S rRNA, was sensitive to the heat degradation.

Sucrose gradient sedimentation of dissociated HeLa cell ribosomes

HeLa cell ribosomes were dissociated into subunits by NaEDTA titration in the presence of 60 mM NaCl. The two subunits which are produced are easily resolved in sucrose gradients, and each subunit has only one type of RNA. 0.5 M NH 4Cl has been used to produce subunits of Escherichia coli ribosomes but under the conditions used here no suitable dissociation was achieved. Several sucrose gradient centrifugation patterns resulting from the exposure of HeLa cell particles to NH 4 + are presented. The removal of particles from 0.5 M NH 4Cl solution into NaEDTA-NaCl produces sedimentation profiles similar to those achieved by NaEDTA-NaCl alone. The stability of ribosomes exposed to several environments was determined by analysis of patterns obtained by sucrose gradient centrifugation. This technique shows that in dissociation buffer Bentonite treated particles are stable to 5 min heating at 50° or less. Ribosomes are also stable to 20 or more h exposure at 4° to pH values below 9.6 while pH 11.0 rapidly destroys any large structure. The partial reduction in size achieved between 9.5 and 11.0 indicates a method for further fractionation of the subunits. Bentonite treated ribosomes in dissociation buffer are very sensitive to attack by externally added ribonuclease. The degree of protection from enzyme attack afforded the RNA in the intact particle was studied by use of sodium dodecyl sarcosinate — an anionic detergent which remains soluble in the cold at high ionic strength. Ribosomal subunits at an RNA concentration of 65 μg/0.1 ml, show altered sedimentation patterns after brief exposure to 2 ng of pancreatic ribonuclease. This result makes the removal of endogenous nuclease a necessary step before further controlled resolution of the components of the subunits can be attempted.