Ribonucleic Acid Preparations Research Articles

Optimized method for RNA extraction from leaves of forest tree species

Extraction of ribonucleic acid (RNA) from woody plants is a difficult task due to the peculiarities of plant material rich in polysaccharides and starch. The available techniques are often ineffective, since they result in the absence/reduced quality/reduced amount of RNA in the final preparation. The method we have optimized is based on the use of cethyltrimethyl ammonium bromide (CTAB), purification by phenol-chloroform extraction, use of lithium chloride and ammonium acetate. The method showed high efficiency for the extraction of RNA from the leaves of birch and poplar samples, in vitro and mature plants, in comparison with previously used methods (extraction using NucleoSpin® RNA Plant (Macherey-Nagel, Germany) columns, Su (2009) method, standard guanidine thiocyanate method). Electropherograms of RNA preparations showed its high integrity and concentration (up to 85 ng/μl), significantly higher purity of the preparation (up to 2.7 times). Purification of the preparation in the process of extraction can significantly reduce the yield of desoxyribonucleic acid (DNA). The optimized method is highly reproducible and can be used for further research, complementary DNA (cDNA) synthesis, qualitative and quantitative PCR analysis. The method allows obtaining high-quality RNA from other objects of agricultural and forest plants.

Distinct degenerative phenotype of articular cartilage from knees with meniscus tear compared to knees with osteoarthritis

To compare the transcriptome of articular cartilage from knees with meniscus tears to knees with end-stage osteoarthritis (OA). Articular cartilage was collected from the non-weight bearing medial intercondylar notch of knees undergoing arthroscopic partial meniscectomy (APM; N=10, 49.7±10.8 years, 50% females) for isolated medial meniscus tears and knees undergoing total knee arthroplasty (TKA; N=10, 66.0±7.6 years, 70% females) due to end-stage OA. Ribonucleic acid (RNA) preparation was subjected to SurePrint G3 human 8×60K RNA microarrays to probe differentially expressed transcripts followed by computational exploration of underlying biological processes. Real-time polymerase chain reaction amplification was performed on selected transcripts to validate microarray data. We observed that 81 transcripts were significantly differentially expressed (45 elevated, 36 repressed) between APM and TKA samples (≥ 2 fold) at a false discovery rate of ≤ 0.05. Among these, CFD, CSN1S1, TSPAN11, CSF1R and CD14 were elevated in the TKA group, while CHI3L2, HILPDA, COL3A1, COL27A1 and FGF2 were highly expressed in APM group. A few long intergenic non-coding RNAs (lincRNAs), small nuclear RNAs (snoRNAs) and antisense RNAs were also differentially expressed between the two groups. Transcripts up-regulated in TKA cartilage were enriched for protein localization and activation, chemical stimulus, immune response, and toll-like receptor signaling pathway. Transcripts up-regulated in APM cartilage were enriched for mesenchymal cell apoptosis, epithelial morphogenesis, canonical glycolysis, extracellular matrix organization, cartilage development, and glucose catabolic process. This study suggests that APM and TKA cartilage express distinct sets of OA transcripts. The gene profile in cartilage from TKA knees represents an end-stage OA whereas in APM knees it is clearly earlier in the degenerative process.

Transcriptional regulation of protein S gene

Background: Protein S (PS) is a known Vitamin K dependent plasma glycoprotein that is produced in many human tissues including the liver. Furthermore, it has an important function in the coagulation cascade as an anticoagulant cofactor when it binds active protein C. The deficiency of PS may lead to the development of deep vein thrombosis. There are many transcription factors which play an important role in the regulation of the transcription of PS. One of these regulatory factors is the liver specific transcription factor hepatocyte nuclear factor 1 (HNF1), a binding site which is located in the 638 base pair promoter area at the 5' end of the PS gene. HNF1 and PS is not well understood and to confirm the importance of HNF1 in regulating expression of PS small interfering ribonucleic acid (siRNA) which will knockout the HNF1 gene expression can be used, and the effect on PS expression monitored. The levels of expression of HNF1 and PS can be determined by real time polymerase chain reaction. Materials and Methods: Preparation of total ribonucleic acid for complementary DNA synthesis, Evaluation the quality of HepG2 ribonucleic acid by agarose gel electrophores is, Real time polymerase chain reaction, Acrylamide gel electrophoresis. Results: HepG2 rRNA bands shows a good quality of RNA expression, Analysis of the total RNA quantity can be determined by spectrophotometric analysis, The best housekeeping genes were UBC and YWHAZ therefore, there has been used as a reference for the primers samples in efficiency test. HNF1 and PROS1 shows a good expression but with low efficiency which is not required to carry further gene knockdown experiment, SYBR green dye is less sensitive than TaqMan because it presents a non specific detection (primer dimers) were all dsDNA products are detected including primer dimers, contaminating DNA, and PCR product from mis annealed primer. Conclusion: The primer pairs are not efficient to carry further gene knockdown experiment and required to design another HNF1 and PROS1 primers. The efficiency of the PCR should be 90%-100% meaning doubling of the amplicon at each cycle. This corresponds to a slope of -3.1 to -3.6 in the Ct vs log template amount standard curve. To obtain accurate and reproducible results, reactions should have efficiency as close to 100% as possible.

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.

Genetic Differences in the Osteogenic Differentiation Potency According to the Classification of Ossification of the Posterior Longitudinal Ligament of the Cervical Spine

We categorized the four types of ossification of the posterior longitudinal ligament (OPLL) of the cervical spine into two groups. We biochemically investigated the genetic differences in the osteogenic differentiation potency between the two groups. To investigate the genetic differences in the osteogenic differentiation potency according to the OPLL classification. Clinical studies on OPLL have revealed that the risk of progression of the ossification area is greatest for continuous and mixed type OPLL. However, until now, these four types of OPLL have been studied as a single condition. We categorized the four types of OPLL into the OPLL continuous (continuous or mixed type) and OPLL segmental groups (segmental or circumscribed type). Paraspinal ligaments were aseptically obtained from OPLL patients during surgery. The fibroblast-like cells that migrated from the explants were used for experiments. The cells were placed in a 60-mm culture dishes for total ribonucleic acid preparation and 12 well microplates for alkaline phosphatase (ALP) activity staining. After cultures reached confluence, the cells were cultured in osteogenic medium. The messenger ribonucleic acid expression of bone morphogenetic protein-2 (BMP-2), osterix, tumor necrosis factor-α-stimulated gene-6, and ALP was analyzed by quantitative real time-polymerase chain reaction. Osteogenic differentiation of fibroblast-like cells was determined by histochemically detecting ALP production. After osteogenic induction, BMP-2 expression increased in the OPLL continuous and segmental groups. Osterix expression increased in the OPLL continuous group only. Tumor necrosis factor-α-stimulated gene-6 expression was suppressed in the OPLL continuous and segmental groups. ALP expression as well as ALP activity staining was higher in the OPLL continuous group than in the OPLL segmental group. CONCLUSION.: The study revealed genetic differences in the osteogenic differentiation potency between the OPLL continuous and segmental groups. We propose to distinguish OPLL continuous group from segmental group in biochemical studies on OPLL.

Use of Non‐Normalized, Non‐Amplified cDNA for 454‐Based RNA Sequencing of Fleshy Melon Fruit

The melon (Cucumis melo L.) fruit is an important crop and model system for the genomic study of both fleshy fruit development and the Cucurbitaceae family. To obtain an accurate representation of the melon fruit transcriptome based on expressed sequence tag (EST) abundance in 454‐pyrosequencing data, we prepared double‐stranded complementary DNA (cDNA) of melon without the usual amplification and normalization steps. A purification step was also included to eliminate small fragments. Complementary DNAs were obtained from 14 individual fruit libraries derived from two genotypes, separated into flesh and peel tissues, and sampled throughout fruit development. Pyrosequencing was performed using Genome Sequencer FLX (GS FLX) technology, resulting in 1,215,359 reads, with mean length of >200 nucleotides. The global digital expression data was validated by comparative reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR) of 40 selected genes and expression patterns were similar for the two methods. The results indicate that high‐quality, nonbiased cDNA for next‐generation sequencing can be prepared from mature, fleshy fruit, which are notorious for difficulties in ribonucleic acid (RNA) preparation.

A relaxin-mediated pathway to preterm premature rupture of the fetal membranes that is independent of infection

OBJECTIVE: This study was designed to show whether the overexpression of relaxin in the decidua of patients with preterm premature rupture of the membranes is independent of or a consequence of chorioamnionitis. STUDY DESIGN: Two experiments were conducted. In the first experiment fetal membranes and decidua were collected from patients with preterm premature rupture of the membranes ( n = 17) or preterm labor ( n = 17) and were divided according to their degree of histologic infection. Messenger ribonucleic acid was isolated from the tissues and quantitative, sequential Northern analyses were carried out for the expression of human relaxin, interleukin-1β, interleukin-6, and interleukin-8. The second experiment was aimed at increasing the numbers of messenger ribonucleic acid preparations in the two extreme categories, uninfected and severely infected tissues, with preterm premature rupture of the membranes and preterm labor. Some samples of messenger ribonucleic acid from the first experiment were rerun with the Northern analyses in the second experiment. These repeat samples showed no statistical differences in the results run at different times. Therefore the data from the respective groups of patients in both experiments were pooled for statistical analysis. RESULTS: In both the first experiment and in the pooled data of the two experiments the expression of the relaxin genes was significantly greater ( P < .005) in the tissues from patients with preterm premature rupture of the membranes compared with those with preterm labor, in the absence of infection. No effect of the level of infection on the expression of relaxin was noted. In contrast, interleukin-6 gene expression was significantly increased ( P < .05) in severely infected tissues, which was independent of whether the delivery was from preterm premature rupture of the membranes or preterm labor. The expression of the interleukin-1β and interleukin-8 genes were only marginally increased even in severe infection. Marked patient variability in expression of the interleukin genes, especially in severe infection, was noted. CONCLUSION: A relaxin-mediated pathway that leads to preterm premature rupture of the membranes may exist independent of infection. (Am J Obstet Gynecol 1998;179:126-34.)

A method for isolation of cytoplasmic RNA from a slime mold, Physarum polycephalum

A procedure for fast and simple preparation of cytoplasmic ribonucleic acid from Physarum polycephalum microplasmodia is described. Microplasmodia are homogenized in a high-magnesium-high-ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid buffer and nuclei are pelleted. The supernatant is extracted with sodium dodecyl sulfate-phenol-chloroform and crude RNA is precipitated. This is further purified by selective ethanol precipitation from 6 m guanidinum hydrochloride. This RNA preparation is suitable for direct use in hybridization studies.

Preparation of 35S-labeled ribonucleic acid and deoxyribonucleic acid and their use for the assay of nucleolytic enzymes (nucleosides and nucleotides. XXXIX.

Radioactive sulfur was introduced into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules by chemical means. The thio group in 4-thiouracil residues, which were introduced into RNA and DNA by sulfhydrolysis, was exchanged with radioactive Na2S via a 4-thiocyanatouracil intermediate. Specific activity of the 35S-labeled RNA and DNA was around 105 cpm/ODU260. The labeled RNA and DNA were successfully applied as substrates for the assay of nucleolytic enzymes or ribonuclease inhibitor. The sensitivity of the assay was 100 times better than that of the current method using non-labeled nucleic acids as the substrate.

Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis.

A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays.

Differential hormonal responsiveness of the ovalbumin gene and its pseudogenes in the chick oviduct.

We describe the isolation of recombinant phages from a chicken gene library which contain two genes designated X and Y. These two genes are linked to the ovalbumin gene (OV) in the order 5'-X-Y-ovalbumin-3' [Royal, A., Garapin, A., Cami, B., Perrin, F., Mandel, J. L., LeMeur, M., Bregegegre, F., Gannon, F, LePennec, J. P., Chambon, P., & Kourilsky, P. (1979) Nature (London) 279, 125-132]. Both genes contain multiple intervening sequences and share limited sequence homology with the authentic ovalbumin gene but are expressed in oviduct cells at different levels. X and Y hybridization probes were prepared in order to study the expression and the relative hormonal responsiveness of these three genes in chicken oviduct. The sequence specificity of the probes was demonstrated by Southern hybridization assays. Northern hybridization studies using the X and Y gene probes indicated the presence of putative precursor molecules in stimulated oviduct ribonucleic acid preparations, which differ in size from those observed for ovalbumin. R0t analysis has demonstrated that, similar to the ovalbumin gene, the level of X and Y gene transcripts is increased by the steroid hormone estrogen, but to varying degrees. The extent of hormonal responsiveness of the three closely related genes is in the order (normalized) relative to ovalbumin of OV:Y:X congruent to 100:10:1. Pulse-labeling studies of these three closely linked genes suggest that in estrogen-stimulated oviduct, the markedly different steady-state levels of the X, Y, and ovalbumin gene transcripts reflect their differential transcription rates.

Effect of detergents on streptolysin S precursor.

Group A streptococci which produce streptolysin S contain a cellular precursor to streptolysin S in the membranes and cytoplasm which is activatable by blending in a Vortex mixer with glass beads and ribonucleic acid (RNA)-core (RNA preparation from yeast). Although no activation of precursor occurred when it was mixed with detergents, it was activated when blended with glass beads and detergents such as Tergitol NP-40 and Brij 35. Maximum activation of precursor was achieved in 1 to 2% detergent, in pH 6.5 buffer, and after 8 min of blending. Detergents Tween 20, 40, 60, and 80, Brij 56, and Lubrol WX also activated precursor, but, of all the hemolysin preparations, those with Tween 40 or 60 or Lubrol WX were the most stable. The addition of RNA-core during or after blending of precursor with detergents enhanced the titer and stability of the hemolysin. This was due in part to the association of the hemolytic moiety with RNA-core. Activation of precursor in the membrane was better with a detergent, whereas that in the cytoplasm was better with RNA-core. Therefore, precursor from two different cellular locations can be differentiated by the effects of RNA-core and detergents on precursor titer.

Liposomes as vehicles for cellular incorporation of biologically active macromolecules.

Lipid vesicles (liposomes) have recently been shown to be a useful vehicle for the delivery of a variety of compounds to cultured cells. Using large unilamellar vesicles composed of phosphatidylserine [LUV(PS)] we were able to encapsulate poliovirus and purified poliovirus ribonucleic acid (RNA) and show that it can be delivered efficiently to cells in an infectious form. LUV-entrapped poliovirus RNA produced infectious titers 100-fold higher than comparable RNA preparations delivered to cells by other techniques. We have made a quantitative analysis of the uptake and infectivity of the vesicle-encapsulated RNA by using various ratios of RNA copies per vesicle and by determining the percentage uptake of labelled lipid and RNA by HeLa cells.

The introduction of poliovirus RNA into cells via lipid vesicles (liposomes)

Large unilamellar vesicles (LUV) composed of phosphatidylserine are capable of encapsulating poliovirus ribonucleic acid (RNA) and delivering it efficiently to cells in an infectious form. The biological activity of vesicle-entrapped poliovirus RNA was 1–2 × 10 4 plaque forming units/nanogram (pfu/ng) and appeared to be enhanced by ribonuclease treatment of the vesicle preparations (infectivity = 1–2 × 10 5 pfu/ng). Vesicle-mediated RNA infection produced equivalent titers in primate and nonprimate cells. Moreover, the data strongly suggest that the ratio of molecules per infectious unit is close to one when the RNA is properly delivered to the cell. A comparative study of LUV and multilamellar vesicles (MLV) indicates that LUV deliver their contents to the cell cytoplasm much more efficiently than MLV. LUV-entrapped poliovirus RNA produced infectious titers 10–100 fold higher than comparable RNA preparations delivered to cells by other techniques.

Lack of protection afforded by ribonucleic acid preparations from Mycobacterium tuberculosis against Mycobacterium leprae infections in mice

Mycobacterial ribonucleic acid preparations from H37Ra, an attenuated strain of Mycobacterium tuberculosis, provide their usual marked protection against M. tuberculosis challenge; however, they provided no protection against Mycobacterium leprae challenge. Suspensions of intact H37Ra were not effective against M. leprae. Suspensions of BCG gave their usual distinct protection against M. leprae challenge.

Preparation of Transfer Ribonucleic Acid from Tobacco Plant and Examination of its Accepting Ability of Acetyl Serine

Amino group of N-terminal serine of tobacco mosaic virus coat protein is known to be blocked by acetyl group, but the mechanism of acetylation has not been clarified. In this report, transfer RNA (tRNA) was prepared from tobacco plant (Nicotiana tabacum xanthi nc) and using this tRNA as an acceptor, whether acetyl serine can be transferred was examined by a competition method. Conclusion that acetyl serine is not transferred by a serine activation system was obtained.

Biochemical studies during the development of the chick embryo. VI. On a deoxyribosyl compound in the ribonucleic acid preparation.

Ribonucleic acid prepared according to modified Kirby’s phenol method from 14 day old chick embryos was fractionated into three peaks by MAK column chromatography. To the peak eluted in the range from 0.65 to 0.7 m NaCl, the name “Peak II” was given tentatively. Purified Peak II was homogeneous upon ultracentrifugation, polyacrylamide gel electrophoresis and elution patterns of chromatographies.The peak gave a positive result with the orcinol reaction. Furthermore, the hydrolyzate by DNase and snake venom was active in promoting the growth of L. leichmannii 7830. The peak was not disrupted by treatment with RNase, but was with DNase. Peak II treated with heat gave the same fractionation profile as the native peak by gel filtration on Sephadex G–200.By treatment with 0.3 n KOH at 37°C for 18 hr, Peak II was resolved into two peaks, Peak II–1 and Peak II–2. It was found that Peak II consisted of the susceptible and the non-susceptible components to the alkali. From the susceptible component (Peak II–2) the ...

Incorporation of Cytokinin N6-Benzyladenine into Tobacco Callus Transfer Ribonucleic Acid and Ribosomal Ribonucleic Acid Preparations

The incorporation of the cytokinin N(6)-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N(6)-benzyladenine-8-(14)C or N(6)-benzyladenine-8-(14)C: benzene-(3)H(G) has been examined. N(6)-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N(6)-benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N(6)-benzyladenosine content of the RNA preparation. The distribution of N(6)-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N(6)-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T(2) and phosphatase, or ribonuclease T(2) and a 3'-nucleotidase. N(6)-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T(2) or phosphatase alone or by successive treatment with ribonuclease T(2) and a 5'-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T(1) and pancreatic ribonuclease converted the N(6)-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N(6)-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.

Evidence for O antigens as the antigenic determinants in "ribosomal" vaccines prepared from Salmonella.

The nature of the protective substance in ribosomal ribonucleic acid and protein extracts of Salmonella has been investigated. The results of experiments in which vaccines were prepared from isogenic strains and strains with defects in lipopolysaccharide synthesis show that O antigens contaminate both ribonucleic acid and protein ribosomal extracts, and are responsible for at least part of their strain-specific protective activity. In addition, it was observed that a ribosomal ribonucleic acid preparation from S. adelaide contains a heat-stable immunogen which is not an O antigen or that gives cross-protection across species lines. The contribution of ribosomes to the immunity induced by "ribosomal vaccines" is discussed.

Cellular streptolysin S-related hemolysins of group A Streptococcus C203S.

Group A streptococci strain C203S, grown in heart infusion broth with 0.3% maltose, produce two cellular hemolysins related to extracellular streptolysin S (SLS). Enzymatic lysis of the streptococci by group C streptococcal phage-associated lysin results in release of low titer, labile hemolysin, which can be stabilized by ribonucleic acid (RNA)-core (RNA preparation from yeast). This labile hemolysin can be detected only after the higher titer cellular streptolysin O is removed by erythrocyte membranes or inactivated by N-ethylmaleimide. The other cellular SLS-related hemolysin is released in a latent state (potential hemolysin) which can be activated to high-titer hemolysin by sonication with RNA-core. The titer of such activated hemolysin depends upon the intensity of sonic energy, duration of sonication, and amount of RNA-core. RNA obtained from the streptococci is far less effective than RNA-core. When the cocci are disrupted by sonication or grinding, potential hemolysin and/or activated form may be released, depending upon the conditions employed. The potential hemolysin material is large and heterogeneous; activation appears to involve, in part, disaggregation or fragmentation. Labile hemolysin, potential hemolysin, and the activated form of potential hemolysin can all be converted to hemolysin having the same hemolytic and physical properties as RNA-core SLS, suggesting that all have the same hemolytic moiety. The presence of glucose in heart infusion broth prevents formation of both potential hemolysin and RNA-core SLS by log-phase cells, whereas addition of glucose to a culture in heart infusion broth with 0.3% maltose stops accumulation of potential hemolysin but does not affect continuation of RNA-core SLS release. These results suggest that potential hemolysin is a cellular precursor to RNA-core SLS.