RNA In Supernatant Research Articles

Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification.

Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications.

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

Background & AimsLiver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.MethodsIntracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.ResultsIn our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.ConclusionsWe provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

Multiply spliced HIV RNA is a predictive measure of virus production ex vivo and in vivo following reversal of HIV latency.

BackgroundOne strategy being pursued to clear latently infected cells that persist in people living with HIV (PLWH) on antiretroviral therapy (ART) is to activate latent HIV infection with a latency reversing agent (LRA). Surrogate markers that accurately measure virus production following an LRA are needed.MethodsWe quantified cell-associated unspliced (US), multiply spliced (MS) and supernatant (SN) HIV RNA by qPCR from total and resting CD4+ T cells isolated from seven PLWH on ART before and after treatment ex vivo with different LRAs, including histone deacetylase inhibitors (HDACi). MS and plasma HIV RNA were also quantified from PLWH on ART (n-11) who received the HDACi panobinostat.FindingsIn total and resting CD4+ T cells from PLWH on ART, detection of US RNA was common while detection of MS RNA was infrequent. Primers used to detect MS RNA, in contrast to US RNA, bound sites of the viral genome that are commonly mutated or deleted in PLWH on ART. Following ex vivo stimulation with LRAs, we identified a strong correlation between the fold change increase in SN and MS RNA, but not the fold change increase in SN and US RNA. In PLWH on ART who received panobinostat, MS RNA was significantly higher in samples with detectable compared to non0detectable plasma HIV RNA.InterpretationFollowing administration of an LRA, quantification of MS RNA is more likely to reflect an increase in virion production and is therefore a better indicator of meaningful latency reversal.FundingNHMRC, NIH DARE collaboratory.

AUTOPHAGY REGULATION BY RUBELLA VIRUS

Aim. Some viruses can subvert host defense mechanism, autophagy, to their own benefit. We analysed the effect of Rubella virus (RV) infection on autophagy in human alveolar epithelial cells A549. Materials and methods. Cells were infected with the wild type and lab-attenuated strain, C-77w and C-77a, respectively, with a multiplicity of infection of 1.0, in parallel, the expression level of genes encoding Beclin1, Atg5, Rab7, and p62 (SQSTM1) proteins participating in different steps of autolysosome formation was measured. To investigate the role of autophagy on RV replication cycle, we measured the amount of infectious RV particles, together with the viral RNA in supernatants and cell lysates, after incubation of A549 cells with wild type or attenuated strain in the presence of the autophagy inhibitor, Bafilomycin A1, or inducer, Rapamycin. Results. The significant increase in Beclin1 and Atg5 gene expression at 24-48 (for the wild type) and 24-72 (for the attenuated type) hours after infection was observed, while significant induction of either Rab7 or SQSTM1 gene expression was not noticed. This effect was correlated with more delayed increase of IFNβ expression and IFNβ-mediated pro-apoptotic gene expression leading to apoptotic cell death 72-96 hours after infection. Moreover, Bafilomycin A1 diminished the RV infection non significantly, as evidenced by the RT-qPCR and plaque assay, while Rapamycin increased the amount of infectious RV particles released by the infected cells more dramatically with wild type comparing with attenuated strain. Conclusion. Thus, we hypothesized that RV can use an antiviral mechanism to prevent degradation and ensure its replication, differentially regulating the process of autophagy, by stimulating the initiation and suppression of later steps.

HIV-1 virological remission lasting more than 12 years after interruption of early antiretroviral therapy in a perinatally infected teenager enrolled in the French ANRS EPF-CO10 paediatric cohort: a case report

Durable HIV-1 remission after interruption of combined antiretroviral therapy (ART) has been reported in some adults who started treatment during primary infection; however, whether long-term remission in vertically infected children is possible was unknown. We report a case of a young adult perinatally infected with HIV-1 with viral remission despite long-term treatment interruption. The patient was identified in the ANRS EPF-CO10 paediatric cohort among 100 children infected with HIV perinatally who started ART before 6 months of age. HIV RNA viral load and CD4 cell counts were monitored from birth. Ultrasensitive HIV RNA, peripheral blood mononuclear cell (PBMC)-associated HIV DNA, HIV-specific T-cell responses (ie, production of cytokines and capacity to suppress HIV infection), reactivation of the CD4 cell reservoir (measured by p24 ELISA and HIV RNA in supernatants upon phytohaemagglutinin activation of purified CD4 cells), and plasma concentrations of antiretroviral drugs were assessed after 10 years of documented control off therapy. The infant was born in 1996 to a woman with uncontrolled HIV-1 viraemia and received zidovudine-based prophylaxis for 6 weeks. HIV RNA and DNA were not detected 3 days and 14 days after birth. HIV DNA was detected at 4 weeks of age. HIV RNA reached 2·17× 10(6) copies per mL at 3 months of age and ART was started. HIV RNA was undetectable 1 month later. ART was discontinued by the family at some point between 5·8 and 6·8 years of age. HIV RNA was undetectable at 6·8 years of age and ART was not resumed. HIV RNA has remained below 50 copies per mL and CD4 cell counts stable through to 18·6 years of age. After 11·5 years of control off treatment, HIV RNA was below 4 copies per mL and HIV DNA was 2·2 log10 copies per 10(6) PBMCs. The HLA genotype showed homozygosity at several loci (A*2301-, B*1503/4101, C*0210/0802, DRB1*1101-, and DQB1*0602-). HIV-specific CD8 T-cell responses and T-cell activation were weak. Findings from this case suggest that long-term HIV-1 remission is possible in perinatally infected children who receive treatment early, with characteristics similar to those reported in adult HIV post-treatment controllers. Further studies are needed to understand the mechanisms associated with HIV remission and whether early treatment of infected children might favour the conditions needed to achieve HIV control after treatment discontinuation. Agence de recherche ANRS (France Recherche Nord & Sud Sida-HIV Hépatites).

Development of a cell culture system based on recombinant hepatitis C virus expressing enhanced green fluorescent protein

Objective To develop a time saving and sensitive cell culture system based on hepatitis C virus chimera expressing enhanced green fluorescent protein(EGFP) and to facilitate the study on HCV pathogenesis and screening of anti-HCV drugs.Methods Enhanced green fluorescent protein reporter gene and a mutation V2440L that can yield higher virus titers were introduced into the C-terminus of non-structural protein 5A (NS5A) of the JFH1 viral genome by using recombinant PCR.The viral RNA was transfected into Huh7.5 cells.Viral RNA in supernatant of HCV RNA-transfected cells was determined after transfection by RT-PCR.HCV replication and infection were determined by immunofluorescence assay.IFN-α was used to evaluate the feasibility of this system for anti-HCV drugs screening.Results The viral RNA replicated efficiently in transfected cells.These cells can produce HCV-EGFP reporter virus.Viral RNA levels in supernatant were 3.06× 105 copies/ml and 7.96×106 copies/ml at 72 h and 9 d after transfection,respectively.The virus titer reached to 104 FFU/ml 9 d after transfection.The expression of EGFP was inhibited by IFN-α in a dose dependent manner in Huh7.5 cells infected by HCV-EGFP reporter virus.Conclusion The recombinant HCV JFH1-EGFP reporter gene system is a time saving,cost effective and sensitive method for studying viral replication cycles and screening of anti-HCV drugs. Key words: HCV; EGFP; Reporter gene; Cell culture

Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo

During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells).In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.

Feline immunodeficiency virus ORF-Ais required for virus particle formation and virus infectivity.

The orf-A (orf-2) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr, vpu, or nef than to the regulatory gene tat encoded by the primate lentiviruses.

Large quantity of ribosomal RNA exists extracellularly in mouse spleen.

When BALB/c mouse spleens were gently homogenized in saline, the resultant supernatant (without cells and tissue debris) contained significant amount of 28S and 18S ribosomal RNA, reaching up to 70% of the total spleen RNA. Haemoglobin assays indicated that less than 15% of the spleen cells were lysed during the homogenization process, indicating that the majority of the spleen 'supernatant RNA' was from the extracellular space of the organ rather than released by the splenocytes as a consequence of grinding. Quantitative RNA analysis showed that the ratio of spleen supernatant RNA/total RNA of BALB/c mice was inversely correlated with age (from approximately 70% at 3 weeks to 45% at 6 months), but that of BXSB mice (an animal model for systemic lupus erythematosus) remained at about 70% irrespective of age. Methyl Green-Pyronin Y staining of paraffin sections of mouse spleen revealed that extracellular RNA was distributed mainly in the sinuses of the organ. Culture supernatants of apoptotic splenocytes contained significant amounts of RNA, suggesting that the extracellular RNA in the spleen might have come from apoptotic lymphocytes. This is supported by the fact that 'thymus supernatant' also contained significant amount of RNA. A possible correlation between spleen extracellular RNA and autoimmune diseases is discussed.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction.

To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. HCV may exist and remain functional in a cultured cell line for a long period.

Ovine aortic smooth muscle cells allow the replication of visna-maedi virus in vitro

Visna-maedi virus induces in sheep an interstitial lung disease characterised by an accumulation of smooth muscle cells (SMC) or myomatosis. Infection by HIV-1 has been recently associated with disorders of the vessel-derived cells: primary pulmonary hypertension, coronary artery disease and smooth muscle tumors in humans. We hypothesized that, besides their regular targets (i.e. macrophages and lymphocytes), lentiviruses could infect smooth muscle cells. Smooth muscle cell cultures derived from ovine aorta were infected with visna-maedi virus strain K1514. The cultured cells were smooth muscle cells as demonstrated by their antigenic expression of alpha-actin and vimentin. The lentiviral infection of the smooth muscle cells was demonstrated by a typical cytopathic effect (syncytia), the expression of virus specific antigens, and the presence of genomic RNA detected by Northern blot analysis and RT PCR. The detection of a reverse transcriptase activity, the presence of viral RNA in supernatants of infected smooth muscle cells detected by RT PCR and their ability to infect ovine permissive fibroblasts demonstrated a productive infection. The ability of smooth muscle cells to be infected by lentiviruses may participate in the pathogenesis of the tissue damage associated with the lentiviruses such as myomatosis in sheep and vascular disease in humans.

An assessment of some methodological criticisms of studies of RNA efflux from isolated nuclei

RNA efflux from isolated nuclei can be studied either as a means of elucidating the general mechanism of nucleo-cytoplasmic RNA transport, or as part of an investigation of the processing and utilization of particular gene transcripts. The present paper describes an assessment of three methodological criticisms of RNA-efflux measurements that are made for the former reason: for such measurements, it is sufficient to show that the post-incubation supernatant RNA is similar overall to homologous cytoplasmic mRNA, rather than to nuclear RNA, that is nevertheless of intranuclear origin, and that alterations to the medium during experiments do not markedly perturb this general nuclear restriction. The results seem to justify the following conclusions. (1) Although degradation of the nuclear RNA occurs during incubation in vitro, this process does not account for the appearance of RNA in the postnuclear supernatant. The degradation can be largely prevented by the addition of serine-proteinase inhibitors without altering the RNA efflux rate. (2) Some adsorption of labelled cytoplasmic RNA to the nuclear surface occurs during both isolation and incubation of the nuclei, and some desorption occurs during incubation. However, these effects introduce errors of less than 10% into the measurements of efflux rates. (3) Exogenous acidic polymers, including polyribonucleotides, disrupt nuclei and increase the apparent RNA efflux rate by causing leakage of nuclear contents. However, this effect can largely be overcome by including the nuclear stabilizers spermidine, Ca2+ and Mn2+ in the medium. In terms of this assessment, it appears that RNA efflux from isolated nuclei in media containing nuclear stabilizers serves as a reasonable model for transport in vivo.

Reverse Transcriptase and 70S RNA in Supernatant From a Human Cell Line2

Reverse Transcriptase and 70S RNA in Supernatant From a Human Cell Line2

Some properties of cytoplasmic RNA fractions of the mouse Ehrlich ascites carcinoma.

Two RNA subfractions from mouse Ehrlich ascites carcinoma 105 000 × g supernatant and three RNA subfractions from the tumor microsomal fraction have been resolved by chromatography on methylated albumin–kieselguhr. The five subfractions have been examined for their amino acid acceptor capacities and nucleotide compositions and by sucrose density-gradient centrifugation. One of the 105 000 × g supernatant fractions has been identified as amino acid acceptor or transfer RNA (tRNA) and the three microsomal components have been identified as tRNA, 5S RNA, and ribosomal RNA. The microsomal tRNA and 5S RNA appear to be associated with ribosomal RNA in a molar ratio approaching 2:1 and together make up about 6–8% of the total microsomal RNA. The second 105 000 × g supernatant RNA fraction has not been identified. It is recovered in widely varying amounts and behaves like heterogeneous material in chromatographic and sedimentation procedures.

Biochemical changes in progressive muscular dystrophy. VII. Studies on the biosynthesis of protein and RNA in various cellular fractions of the muscle of normal and dystrophic mice

Protein nitrogen content, RNA concentration, and in vivo incorporation of L-[U-14C]leucine into protein and of [2-14C]uridine into RNA of homogenate and various fractions of muscle of normal and dystrophic mice were measured at various stages of the disease. Protein nitrogen content was always lower in dystrophic than in normal muscle, and this became more pronounced with the progress of the disease. Most of the decrease was due to loss of proteins from the myofibrils. RNA content increased in the homogenate, nuclei–myofibrils, supernatant, and microsomes of dystrophic muscle. In the mitochondria of dystrophic muscle, no change was noted compared to controls. The ratio of RNA content to protein in the homogenate, nuclei–myofibrils, supernatant, and microsomes was also greater in dystrophic muscle. It was not changed in dystrophic muscle mitochondria. Incorporation of L-[U-14C]leucine into proteins of dystrophic muscle homogenate and various fractions also increased to variable degrees over that in the controls. It was further observed that mitochondrial and microsomal protein incorporate L-[U-14C]leucine in dystrophic muscle at an increased rate but the disappearance of 14C was even greater, compared to controls.In vivo incorporation of [2-14C]uridine into RNA of dystrophic muscle increased at 30 days', remained the same at 60 days', and declined at 90 days' duration of the disease. Similar results were also obtained in the nuclei–myofibrillar fraction of dystrophic muscle. In all other fractions an increase was noted in incorporation in dystrophic muscle. The incorporation of [2-14C]uridine into RNA in supernatant and microsomes was higher in dystrophic muscle but the disappearance of 14C was greater, compared to controls. It is quite evident in the microsomal fraction at 90 days, where no change in the incorporation is noted in normal and dystrophic animals.

Some Effects of 2,4-Dichlorophenoxyacetic Acid and Sucrose on Orotic-Acid Uptake in Nucleic Acids of Excised Cotton Cotyledons

The nucleic-acid metabolism of excised cotton cotyledons floated on Hoagland nutrient medium was studied. The effects of 2,4-D and sucrose on the total level of nucleic acid maintained in the supernatant RNA and particulate nucleic acid and the rates of orotic acid-6-C14 incorporation into the nucleic acids during a 4-hr period after various periods of culture were determined. Sucrose stimulated a loss of nucleic acid in the supernatant RNA and stimulated an increase of particulate nucleic acid during culture. Glucose and fructose in the culture medium were equally as effective as sucrose. Mannitol was not as effective, showing that the sugar effect was probably not a consequence of osmotic properties of the culture medium. The rates of incorporation of orotic acid-6-C14 into supernatant RNA increased rapidly in untreated cotyledons during culture. At the same time, there occurred a decrease in nucleic acid of this fraction and an increase in particulate nucleic acid. 2,4-D, at a growth-inhibiting concent...

The fractionation and characterization of nuclear ribonucleic acid from rat liver.

RNA isolated from liver nuclei was fractionated by sucrose gradient centrifugation. 4 fractions of RNA (> 40 S, 33 S, 19 S and 6 S) were obtained, and these RNA's were characterized with respect to absorption spectrum, changes in extinction at 260 and 280 mμ on alkaline hydrolysis, changes in extinction at 260 mμ on heating, and kinetics of digestion by RNAase. Microsomal, cytoplasmic supernatant, and whole tissue RNA's were similarly fractionated and many of the values for the above determinations were obtained on the resulting fractions. Nuclear RNA appears to have the following components: (a) high-molecular-weight (33 S and 19 S) ribosomal-like RNA, (b) low-molecular-weight (6 S) RNA, whose physical properties are distinct from cytoplasmic supernatant RNA, (c) a group of RNA's with heterogeneous sedimentation properties, which may correspond to messenger RNA and/or ribosomal precursors. Possible relationships of various nuclear RNA's to nuclear function are discussed.

The fractionation and characterization of nuclear ribonucleic acid from rat liver

RNA isolated from liver nuclei was fractionated by sucrose gradient centrifugation. 4 fractions of RNA (> 40 S, 33 S, 19 S and 6 S) were obtained, and these RNA's were characterized with respect to absorption spectrum, changes in extinction at 260 and 280 mμ on alkaline hydrolysis, changes in extinction at 260 mμ on heating, and kinetics of digestion by RNAase. Microsomal, cytoplasmic supernatant, and whole tissue RNA's were similarly fractionated and many of the values for the above determinations were obtained on the resulting fractions. Nuclear RNA appears to have the following components: (a) high-molecular-weight (33 S and 19 S) ribosomal-like RNA, (b) low-molecular-weight (6 S) RNA, whose physical properties are distinct from cytoplasmic supernatant RNA, (c) a group of RNA's with heterogeneous sedimentation properties, which may correspond to messenger RNA and/or ribosomal precursors. Possible relationships of various nuclear RNA's to nuclear function are discussed.

The nucleotide composition of ribonucleic acids from subcellular components of yeast, Escherichia coli and rat liver, with special reference to the occurrence of pseudouridylic acid in soluble ribonucleic acid

The analyses of the nucleotide composition of RNA's derived from various subcellular fractions of yeast and Escherichia coli B, revealed that the RNA found in the supernatant fraction has a unique nucleotide composition as compared with the RNA from the particle fractions. The supernatant RNA is characterized by the presence of a considerable amount of pseudouridylic acid and by higher cytidylic acid and lower adenylic and uridylic acid contents. Predominant localization of pseudouridylic acid in the RNA of the “pH 5 enzyme fraction” of rat liver was also observed. The relation between the ability to bind [ 14C]leucine and the content of pseudouridylic acid in RNA's prepared from large granules, small granules, and some subfractions of the 105,000 × g supernatant of yeast cells has also been investigated. The RNA prepared from the supernatant fraction is the only active RNA, those prepared from particle fractions failing to incorporate [ 14C]leucine. In five preparations of RNA derived from the acid-fractionated supernatant subfractions and the unfractionated supernatant, it was found that the ability to incorporate [ 14C]leucine into different RNA's is directly proportional to their pseudouridylic acid content. These data suggest that this nucleotide is characteristic of the RNA which incorporates amino acids (at least leucine).

The concentration and collision frequency of microsomal particles and supernatant RNA in rabbit reticulocytes

An attempt has been made to evaluate the collision frequency between the microsomal particles and the supernatant ribonucleic acid (S-RNA) molecules in rabbit reticulocytes. This has been done in order to examine the theory that S-RNA carries the activated amino acids to the microsomal particles in hemoglobin synthesis. The concentration of particles and S-RNA in the cell was found to be 3 × 10 −5 and 6 × 10 −5 M, respectively. The collision frequency has been calculated from Smoluchowski's equation. The results indicate that if more than 0.01% of the collisions result in amino acid transfer, then the collision frequency is sufficient to support the rate of peptidebond synthesis which is actually observed.