Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis.In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg RNA viruses have extreme mutation frequencies, due in large part to the erroneous nature of the viral RNA dependent RNA polymerases (RdRp) that replicate their genomes. Since RdRp lack proofreading and repair mechanisms, the use of base analogs as RNA mutagens to increase lethal mutations and extinguish the virus population is a promising antiviral strategy. Recently, a screen for resistance to this antiviral treatment identified a higher fidelity RdRp variant of poliovirus, indicating that RdRp fidelity can be modulated by single amino acid substitutions. To extend these observations to other viruses, we performed a similar screen using Coxsackie virus B3 (CVB3). We identified a new high fidelity RdRp variant which was also resistant to amiloride compounds that have no known mutagenic activity. Using wild type and RdRp fidelity variants of poliovirus and CVB3, we show that amiloride compounds do have mutagenic activity and act on RNA virus populations indirectly, by altering intracellular ion concentrations that affect polymerase fidelity. Our results identify a new means of targeting viruses through increases in mutation frequency using non-nucleoside compounds that alter the cellular environment in which the virus replicates. The mechanism for this resistance remains unclear.Amiloride and its derivatives are potassium-sparing diuretics used to treat hypertension and to prevent hypokalemia associated with congestive heart failure. These compounds act by inhibiting epithelial NaWe isolated one of these same CVB3 RdRp mutants in a screen for resistance to RNA mutagens with the goal of identifying higher fidelity RdRp variants. RNA mutagens such as ribavirin, 5-fluorouracil and 5-azacytidine are base analogs that are incorrectly inserted into the genome by the RdRp during replication and result in the accumulation of lethal mutations over several passages, a process referred to as lethal mutagenesis6 TCID50) in 50 µM of either mutagen. Every five passages, the viral RNA-dependent RNA polymerase (RdRp) region was sequenced (4 nucleotides (P = 0.0225), thereby confirming the higher fidelity of this variant. As expected, treatment with 400 µM ribavirin significantly increased the mutation frequency of both virus populations (P<0.0001); however, the mutation frequency of A372V remained lower than wild type (P = 0.039). Furthermore, individual clones in the wild type virus population more often presented multiple mutations compared to A372V . Mutagen concentrations above 100 µM decreased viral viability by over 90% . In ordeequenced . Wild tyequenced . A similequenced . The untleotides . The A37to A372V . These rd primer  [21]. Byd primer , confirmCuriously, A372V was previously isolated in a screen for resistance to amiloride, a compound with no known mutagenic activity, that was shown to reduce CVB3 titers by partially inhibiting RNA synthesis 4 nucleotides (P = 0.0005). Similarly, A372V virus also increased mutation frequency, from 2.5 to 6.2 mutations per 104 nucleotides (P = 0.0008). However, since the basal mutation frequency of A372V was lower, this increase was better tolerated, explaining the high virus titers observed (4 nucleotides to 8.5 for EIPA (P = 0.0028) and to 9.9 for MIA (P<0.0001). Although a tendency towards increase was observed for benzamil (6.3 mutations per 104 nucleotides), no statistically significant difference was established (P = 0.105). Furthermore, treatment of A372V with EIPA also increased the mutation frequency from 2.5 to 4.1 mutations per 104 nucleotides (P = 0.05) and correlated the higher titers compared to wild type virus Since it is unlikely that the same mutation confers resistance to two unrelated antiviral mechanisms, we explored whether amiloride has a previously unknown mutagenic activity. This possibility was not evident, since amiloride is not a base analog such as ribavirin, AZC and FU, whose mutagenic effects result from their direct misincorporation into genomes by the error-prone RdRp. We hypothesized that in addition to inhibiting RNA replication, amiloride compounds increase the virus mutation frequencies of the RNA genomes that are replicated. To address this, wild type and A372V virus infections were performed in the presence of either ribavirin or amiloride, or in the absence of either compound and the mutation frequencies of the resulting populations were determined . At 400 observed . Next, wobserved . Again, 4 nucleotides sequenced and increased to 8.2 mutations per 104 nucleotides (P = 0.039) upon amiloride treatment. The high fidelity G64S population had a basal mutation frequency of 4.0 mutations per 104 nucleotides that increased to 6.7 mutations per 104 nucleotides (P = 0.021) in amiloride, but remained significantly lower than wild type virus (P = 0.05). In order to determine whether lower basal mutation frequencies correlated with resistance, amiloride treated and untreated populations were titrated (P = 0.006). Our results therefore confirm for two different high fidelity RdRps, that increased fidelity and lower mutation frequency confer resistance to amiloride.The G64S variant of poliovirus is another higher fidelity RdRp variant whose fidelity altering determinant maps to a different region of the RdRp . Previoutitrated . A higheet al. isolated a second RdRp variant, S299T, in their screen for amiloride resistance Along with A372V, Harrison 4 nucleotides, P = 0.0034), suggesting that this variant encodes a lower fidelity polymerase. Indeed, treatment with 400 µM ribavirin increased the mutation frequency to 28.1 mutations per 104 nucleotides (P<0.0001), significantly higher than wild type (P = 0.0207) and A372V (P<0.0001) , suggesting that S299T polymerase is completely unaffected by the mechanism by which amiloride increases mutation in wild type and A372V viruses. Hence, while A372V resists the mutagenic activity of amiloride by increasing RdRp fidelity; S299T partially resists RNA replication inhibition and this mutagenic effect entirely.Finally, to examine the potential effects of the S299T mutation on RdRp fidelity, infections were performed in the presence of ribavirin, amiloride, EIPA, or under mock-treatment and mutation frequencies were determined for each population. Unlike A372V, the mutation frequency of S299T did not suggest higher fidelity compared to wild type . Rather,<0.0001)  and in aOur results argue that increases in mutation frequency are a previously unknown antiviral mechanism of amiloride compounds, but the relative contribution of RNA synthesis inhibition and RNA mutagenesis is unclear. To examine the relative contribution of mutagenesis to the overall antiviral effect, we determined the mutation frequencies of wild type CVB3 virus populations treated with increasing concentrations of ribavirin or amiloride. These data were used to determine the fold increase in mutation frequency of wild type virus at increasing concentrations of drug compared to untreated control. Indeed, ribavirin treatment showed a dose-dependent increase in mutation frequency . These r+, Ca2+, Mg2+, which in turn can alter the equilibrium levels of other ions, including Mn2+2+ and Mn2+ are interesting candidates since they are cofactors essential for incorporation activity of polymerases such as viral RdRp. In our own cell culture conditions, we confirmed that in addition to the more commonly studied effects on Na+, Mg2+ levels are also significantly perturbed in cells treated with amiloride (P = 0.0009)  .2, MgCl2 or MnCl2 and the resulting mutation frequencies were determined. Growth of all three viruses in either NaCl or CaCl2 had no significant effect on mutation frequency  as would be expected of the higher fidelity variant. In contrast, the mutation frequency of S299T remained unchanged  and fixed (passage 7 onward) the same high fidelity A372V mutation that was selected by RNA mutagen  which could in turn affect the equilibrium of other ions, such as Mn2+2+ concentrations, although 800 µM amiloride treatment was necessary because of detection limits  or indirectly through effects on Na+ channels and exchangers will be difficult to determine. Studies with other channel blockers of different structure may help to pinpoint this mechanism, although redundancy between channels may mask their effect. Nevertheless, the mutagenesis and resistance profiles of the A372V and S299T amiloride-resistant variants support this link between cation concentrations and amiloride's mutagenic effect.What is the molecular basis for amiloride induced mutagenesis? The lack of dose dependence of the observed mutagenic effect at higher concentrations of amiloride  that is n limits . Since Mrequency . Whether2+ and Mn2+ but in all cases, the frequencies were significantly lower than wild type populations under the same treatments. Unlike S299T, this variant is solely resistant to the mutagenic antiviral activity since its RNA synthesis is as inhibited by amiloride as wild type virus , resulted in the selection of the high fidelity A372V variant over S299T  and Vero cells were maintained in DMEM medium with 10% newborn calf serum. Plasmid bearing the cDNA of Coxsackie virus B3 (Nancy) strain was a kind gift of F. van Kuppeveld . Plasmids containing poliovirus cDNAs were previously described Ribavirin IUPAC 1--1H-1,2,4-triazole-3-carboxamide);5-fluorouracil IUPAC 5-fluoro-1H-pyrimidine-2,4-dione;5-Azacitidine IUPAC 4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one;Amiloride IUPAC 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazine-2-carboxamide;EIPA IUPAC 3-amino-6-chloro-N-(diaminomethylidene)-5-[ethyl(propan-2-yl) amino]pyrazine-2-carboxamide;MIA IUPAC 3-amino-5-[tert-butyl(methyl)amino]-6-chloro-N-(diaminomethylidene)pyrazine-2-carboxamide;benzamil IUPAC 3,5-diamino-N-(N'-benzylcarbamimidoyl)-6-chloropyrazine-2-carboxamide.Sal I and poliovirus cDNA plasmids, with Eco RI. Linearized plasmids were purified with the Qiagen PCR purification kit. 2.5 µg of linearized plasmid was in vitro transcribed using T7 RNA polymerase (Fermentas). 8 µg of transcript was electroporated into 4×106 Vero cells that were washed twice in PBS (w/o Ca2+ and Mg2+) and resuspended in PBS (w/o Ca and Mg) at 107 cells/ml. Electroporation conditions were as follows: 0.4 mm cuvette, 25 µF, 700 V, maximum resistance, exponential decay in a Biorad GenePulser XCell electroporator. Cells were recovered in DMEM. 500 µl of p0 virus stock was used to infect 3×106 Vero cells in T25 flasks, to produce p1 virus. 250 µl of p1 virus was used to infect 1×107 Vero cells in DMEM-10% NCS in T75 flasks, to produce p2 virus. For each passage, virus was harvested at total cytopathic effect (CPE) by one freeze-thaw cycle and clarified by spinning at 10 K rpm for 10 minutes.All studies were performed on virus stocks  generated from cDNA infectious clones. The A372V and S299T variants were constructed using the Quikchange XL site directed mutagenesis kit (Stratagene) and the CVB3-Nancy infectious cDNA. Three infectious cDNA clones of each variant (A372V and S299T) were obtained, sequenced and used to generate three independent virus stocks. Three virus stocks of wild type virus were also prepared in this manner. Each of three stocks served as one of three triplicate samples in replication studies, and RNA mutagen and amiloride compound sensitivity assays. For mutation frequency data, the virus population generated by clone 1 for each variant was used throughout the study. Basal mutation frequencies were determined on 2 independently generated samples of each variant and no statistically significant differences in mutation frequencies were found. CVB3 cDNA plasmids were linearized with 4 Vero cells plated in 100 µl of DMEM. After 5 days living cell monolayers were colored by crystal violet. TCID50 values were determined by the Reed and Muensch method. By plaque assay. HeLa cells were seeded into 6-well plates and virus preparations were serially diluted (10-fold) in PBS. Cells were washed twice with PBS and infected with 250 µl of dilution for 30 minutes at 37°C, after which a semisolid overlay comprised of DMEM medium and 1.2% w/v Avicell (FMC Biopolymer) was added. 2 days after infection, cells were washed and stained with crystal violet 0.2%, and plaques were enumerated.Ten-fold serial dilutions of virus were prepared in 96-well round-bottom plates in PBS. Dilutions were performed in octuplate and 100 µl of dilution were transferred to 102 or 1 mM MnCl2 for 2 hours, then infected with 106 TCID50 of CVB3. Blind serial passages were then performed on fresh mutagen-treated HeLa cell monolayers (250 µl of virus-containing supernatant). At the indicated passage intervals, viral RNA was extracted from purified virions with Trizol reagent (Invitrogen) and RT-PCR was performed . PCR products were sequenced to identify consensus sequence changes within the RdRp region, between nucleotides 4701 and 7903.HeLa cell monolayers were pretreated with 50 µM ribavirin, 50 µM 5-Azacytidine, 5 mM MgCl2, or MnCl2) or 10 hours (amiloride compounds) with different concentrations of compound as indicated. We chose and verified concentrations of compounds that were not toxic to cells over a 72 hours period. For amiloride compounds, we chose and confirmed concentrations corresponding to virus inhibitory concentration (IC50) values that were not toxic to cells, as determined by Harrison et al.50 or plaque assay) were determined.HeLa cell monolayers in 6-well plates were pretreated for 2 hours  and purified. 5 µg of total RNA were used per sample (measured by Nanodrop). Gels were transferred onto a nitrocellulose membrane (Whatman Turboblotter SuperCharge Nylon membrane kit), hybridized overnight with a dCTP-α32P labeled DNA probe corresponding to 200 bp of the RdRp, visualized on a Storm Phosphorimager and analyzed by ImageQuant.For replication studies, HeLa cells were either pretreated with 400 µM amiloride or mock treated and infected at an MOI of 10. For one-step growth kinetics, transfected cells were frozen at different time points after infection and later titered by TCID4 nucleotides sequenced was determined using the total mutations identified per population over the total number of nucleotides sequenced for that population multiplied by 104. For each population, between 68 and 178 clones were sequenced representing between 58,000 to 153,000 nucleotides per sample  and TopoTA cloned (Invitrogen). Blue/white screening was used on single colony transformants, positive clones were sequenced (GATC Biotech). Sequence data was analyzed using the Lasergene software package (DNAStar Inc). For statistical purposes, we retained sequence data only over the region for which every clone was represented (859 nucleotides for CVB3 and 884 nucleotides for PV). The number of mutations per 10mple see . If the 2+, before lysis in H2O-Tween 0.1%. The ratiometric magnesium indicator mag-fura-2 (Invitrogen) was added to a final concentration of 2 µM to 1 ml of sample and fluorescence was measured at 25°C using a Quanta-Master QM4CW spectrofluorometer (PTI) using a 1 cm path length quartz cuvette thermostated at 25°C. Excitation scans were performed from 250 to 490 nm with 1 nm steps, using 1 nm bandwidth; emission was monitored at 530 nm with 5 nm bandwidth. Continuous recordings of fluorescent intensities at 330 nm and 370 nm were transformed into 330/370 wavelength ratios.A confluent layer of HeLa cells was treated with 800 µM amiloride for 10 hours and incubated with PBS containing 100 µM Ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for 15 min, to chelate Ca2+concentration assays, the two-tailed paired student's t tests were used to determine significance with 95% confidence intervals. For mutation frequencies, χ2 tests and two-tailed Mann Whitney U tests were performed. χ2 tests compared the total number of mutations in a population to total nucleotides sequenced. Mann Whitney tests compared the ranked scores of number of mutations found in individual clones grouped by population. In all cases, Mann Whitney tests gave the more conservative P values and are the indicated here. Statistics were performed using Prism software (GraphPad Inc).For one step growth curves, mutagen treatment assays and Mg32P]-ATP (7000 Ci/mmol) was from MP Biomedical; T4 polynucleotide kinase was from USB; ATP and GTP were from GE Healthcare; all other reagents were of the highest grade available from Sigma, Fisher or VWR. RNA oligonucleotides were purified by denaturing PAGE and end-labeled by using [γ-32P]ATP and T4 polynucleotide kinase as described previouslyAll RNA oligonucleotides were from Dharmacon Research, Inc.; -primer-template substrate in 50 mM HEPES pH 7.5, 5 mM MgCl2, 60 µM ZnCl2 and 10 mM 2-mercaptoethanol. Reactions were initiated by the addition of ATP or GTP (1 or 5 mM) in 50 mM HEPES pH 7.5, 5 mM MgCl2, 60 µM ZnCl2, 10 mM 2-mercaptoethanol and 200 mM NaCl. Reactions were quenched at various times by addition of quench buffer . CVB3 RdRp was diluted immediately prior to use in 50 mM HEPES pH 7.5, 20% glycerol and 10 mM 2-mercaptoethanol. The volume of enzyme added to any reaction was always less than or equal to one-tenth the total volume. Products were resolved by denaturing PAGE. Stopped-flow fluorescence assay: Pre-steady state stopped-flow fluorescence experiments were performed using a Model SF-2001 stopped-flow apparatus  equipped with a waterbath. All reactions were performed at 30°C. 2 µM CVB3 3Dpol was mixed with 0.5 µM primer-template substrate containing 2-aminopurine ribonucleoside on the 5′ side of the templating base2, 60 µM ZnCl2 and 10 mM 2-mercaptoethanol. Reactions were initiated by the addition of 1 mM ATP in 50 mM HEPES pH 7.5, 5 mM MgCl2, 60 µM ZnCl2, 10 mM 2-mercaptoethanol and 200 mM NaCl. After mixing, reactant concentrations were reduced by 50%. Fluorescence emission was monitored by using a 370 nm cut-on filter . The excitation wavelength used was 313 nm. For each experiment, at least four fluorescence traces were averaged. The relative fluorescence was plotted as a function of time and fit to a single exponential equation, Reactions were performed at 30°C. Quenched reaction mixtures were heated to 70°C for 2–5 min prior to loading 5 µl on a 20% denaturing polyacrylamide gel containing 1X TBE and 7 M urea. Electrophoresis was performed in 1X TBE at 85 watts. Gels were visualized by using a PhosphorImager and quantified by using the ImageQuant software (Molecular Dynamics). Data were fit by nonlinear regression using the program, KaleidaGraph .Figure S12+ concentrations. HeLa cells were treated for 10 hours with 800 µM or without amiloride, washed, and lysed. Increases in intracellular Mg2+ concentrations were determined by the change in the ratio of emission signals at 330/370 nm using mag-fura-2 indicator. The mean values and S.E.M. are shown. * P = <0.05, N = 6.Amiloride treatment of cells increases free intracellular Mg(0.39 MB EPS)Click here for additional data file.Figure S2Localization of fidelity altering mutations in picornavirus RdRp. The crystal structure of the CVB3 RdRp (3.00 MB EPS)Click here for additional data file.Figure S32 in the media. One-step growth kinetics of wild type A372V and S299T viruses were performed in the presence of 5 mM MgCl2. HeLa cells were infected at MOI of 10 and the progeny virus was quantified at different hours after infection by TCID50 assay. Mean titers (TCID50/ml) ± S.E.M are shown, N = 3, no significant difference found.Replication of wild type, A372V and S299T is not affected by increasing the concentration of MgCl(0.50 MB EPS)Click here for additional data file.Table S1Mutation distribution data for experiments presented.(0.08 MB PDF)Click here for additional data file.


Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production.The  Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus  Neurospora the production of one type of small RNA called qiRNA, which is specifically induced after DNA damage, requires the RdRP QDE-1. Here, we investigated the precise contributions of QDE-1 to the synthesis of ssRNA and dsRNA. We show that QDE-1 is surprisingly promiscuous in its template choice in that it is able to synthesize RNA from both ssRNA and single-stranded DNA (ssDNA). These results suggest that QDE-1 first generates ssRNA from a DNA template and then converts the ssRNA into dsRNA; this combination of activities in one protein ensures the specific action by RdRP on aberrant RNA in lieu of other single-stranded cellular RNA. In addition, we identified Replication Protein A, a ssDNA-binding protein that interacts with QDE-1, as an essential factor for small RNA production. Furthermore, we were able to reconstitute synthesis of dsRNA from ssDNA in a test tube using purified QDE-1 and RPA proteins, demonstrating the ability of this relatively simple biosynthetic system to generate the nucleic acid trigger for gene regulation. Together, these results uncover the details of a new and important small RNA production mechanism in cells.Small RNA molecules (20–30 nucleotides) play important roles in many cellular processes in eukaryotic organisms by silencing gene expression. To generate the many forms of small RNAs, DNA is first transcribed to produce single-stranded RNA (ssRNA), which then is converted to double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (RdRP). However, it is not clear how the ssRNA templates are synthesized from DNA and specifically recognized by RdRPs amidst a sea of single-stranded, cellular RNAs. We previously showed that in the filamentous fungus  RNA interference (RNAi) refers to a group of post-transcriptional or transcriptional gene silencing mechanisms conserved from fungi to mammals Caenorhabditis elegans, the production of endogenous dsRNA and siRNA requires RNA-dependent RNA polymerases (RdRPs), which can generate dsRNA using single-stranded RNAs (ssRNAs) as templates In fungi, plants, and In plants, the DNA-dependent RNA polymerase IV (Pol IV) is important for siRNA production and RNAi-directed transcriptional silencing Neurospora crassa, quelling is a post-transcriptional silencing mechanism triggered by multiple copies of transgenes during vegetative growth In the filamentous fungus qde-2, as well as other RNAi and putative antiviral genes Neurospora, and their production depends on QDE-1, QDE-3, and Dicers. qiRNA biogenesis also requires the DNA damage-induced aRNA from the rDNA locus. In qde-1 and qde-3 mutants, the induction of rDNA-specific aRNA by DNA damage is abolished, indicating their essential roles in aRNA production. Surprisingly, partially purified RdRP QDE-1 can generate RNA from single-stranded DNA (ssDNA) in vitro, suggesting that QDE-1 is also a DdRP that generates aRNA and then converts it into dsRNA using its RdRP activity.We previously discovered that dsRNA, but not siRNA, activates the transcription of In this study, we demonstrate that QDE-1 is indeed a bona fide DNA-dependent RNA polymerase: recombinant QDE-1 displays DdRP activity that is much more robust than its RdRP activity. In addition, we further investigate the mechanism of aRNA and dsRNA production after DNA damage. Our genetic and biochemical results support a model in which QDE-1 is recruited by ssDNA-binding protein Replication Protein A (RPA) and the RecQ DNA helicase QDE-3. QDE-1 first acts as a DdRP to produce ssRNA and then as an RdRP to convert the ssRNA into dsRNA, a process that is strongly promoted by RPA. These results suggest a mechanism for the generation of aRNA and provide a potential explanation for how aRNA is specifically recognized by RdRPs.Neurospora exhibits both RdRP and DdRP activities Saccharomyces cerevisiae to near homogeneity  was mutated to alanine (QDE-1ΔNDA) The crystal structure of QDE-1 has shown that its catalytic core is structurally similar to eukaryotic DNA-dependent RNA polymerases ogeneity  [8]. Botogeneity , but due32P-labeled RNA products and shifted the ethidium bromide stained DNA band to template length. RQ1 DNase, which degrades DNA, shifted the majority of the labeled RNA products to the template length. RNase V1, which cleaves base-paired DNA or RNA, degraded the product completely. In contrast, RNase A, which degrades RNA with single-stranded character, did not affect the reaction products. These results indicate that the products of DdRP activity of QDE-1 are mostly ssRNA hybridized to its template as DNA/RNA hybrids. As expected, the RdRP products of QDE-1ΔN were dsRNA, as neither RQ1 DNase nor RNase H had any effect on the products, while RNase V1 treatment resulted in their complete degradation . Similarly, Myc-QDE-1 purified from th dsDNA .If QDE-1 functions as the RNA polymerase that generates the aberrant transcripts in vivo, it needs to be recruited to ssDNA where the aRNAs are expressed. Previously, QDE-1 was found to interact with Replication Protein A (RPA) rpa mutants in Neurospora. The knock-outs of rpa-1 and rpa-2 are lethal, but the knock-out of rpa-3, the smallest subunit of RPA, is viable and does not cause severe growth and developmental defects. To obtain rpa-1 and rpa-2 knock-down strains, we introduced constructs that can express an inverted repeat specific for rpa-1 (dsrpa-1) or rpa-2 (dsrpa-2) into a wild-type strain. We first measured the induction of QDE-2 by DNA damage agent histidine, an indication of endogenous dsRNA generation korpa-3 mutants, and the basal levels of QDE-2 in the korpa-3 mutants were lower than the basal wild-type level. Also, in the dsrpa-1 and dsrpa-2 knockdown strains, the induction of QDE-2 by histidine was mostly abolished. In addition, DNA damage-induced qiRNA and aRNA expression at the rDNA region were both eliminated in the korpa-3 mutant . As shown in There are two types of products made by QDE-1 using these circular ssDNA templates as indicated by the prominent high molecular weight bands and the low molecular weight smear . The priE. coli but failed to detect a clear effect on QDE-1 activity (unpublished data). This suggests that the effect of RPA on QDE-1 activity extends beyond its ssDNA binding character.Although QDE-1 can produce some dsRNA from ssDNA, the majority of the QDE-1 products are DNA/RNA hybrids, which prevent the generation of dsRNA. Thus, for robust dsRNA production, a mechanism should exist to unwind the nascent DNA/RNA hybrids or to prevent their formation. Since RPA binds to ssDNA, we examined its effect on QDE-1 RNA polymerase activity. In addition, we were interested to know whether QDE-1 can use RPA-bound ssDNA as a template. Different concentrations of human RPA complex were incubated with ssDNA (a 175 nt template) before the addition of recombinant full-length QDE-1. The reaction products were separated by a denaturing polyacrylamide gel. As shown in Drosophila Dicer-2 purified from Sf9 insect cells). As shown in It was previously shown that for ssRNA templates in vitro, QDE-1 can utilize the 3′ ends of ssRNAs to create dsRNAs in which the complementary strands are covalently attached to each other, as a result of a mode of initiation called back-priming RPA participates in many DNA metabolic processes under normal growth conditions, but qiRNA and rDNA-specific aRNA are preferentially produced after treatments with DNA damaging agents, suggesting that the interaction between QDE-1 and RPA requires additional determinant(s). QDE-3 and its eukaryotic homologs such as human BLM and WRN and yeast SGS1 play important roles in DNA repair, genome maintenance, and DNA replication Neurospora. c-Myc-tagged QDE-3 and FLAG-tagged RPA-1 constructs under the control of the Neurospora quinic acid inducible promoter (a rather weak promoter) were co-transformed into a wild-type Neurospora strain. As shown in Neurospora strain  QDE-1, but not the canonical RNA polymerases, is required for aRNA production from the rDNA locus after treatment with DNA damaging agents Neurospora exhibit robust DdRP activity using ssDNA but not dsDNA templates, and the DdRP activity of QDE-1 is considerably higher than its RdRP activity. (iii) QDE-1 can directly generate dsRNA from ssDNA templates. (iv) RPA, a QDE-1-interacting protein that binds to ssDNA, is required for aRNA and qiRNA production and strongly enhances dsRNA synthesis. Therefore, although we cannot exclude the possibility that other RNA polymerases also contribute to the rDNA specific aRNA production, the evidence suggests that QDE-1 should be a major RNA polymerase involved in this process. Although it was previously shown that QDE-1 can produce some small RNAs from ssRNA in vitro The genetic and biochemical results presented here and in previous studies Neurospora, QDE-3 and RPA are responsible for the generation of ssDNA template for QDE-1. Furthermore, the loss of QDE-1-RPA interaction in the koqde-3 mutant suggests that QDE-3 contributes to this interaction. The involvement of QDE-3 in this process suggests that although RPA is involved in many processes of DNA metabolism and ssDNA is generated during normal DNA replication, QDE-1 is only recruited to the ssDNA locus if both RPA and QDE-3 are present. This conclusion is supported by the observation that qiRNA and aRNA production is abolished in the koqde-3 mutant Our results suggest that the generation of ssDNA after DNA damage/replication stress is a trigger for the production of aRNA and small RNA. The requirement of RPA in aRNA and qiRNA production and the interaction between RPA and QDE-1 support this model and suggest that QDE-1 may be recruited to ssDNA template by RPA. In addition, like their mammalian homologs, RPA and QDE-3 apparently associate with each other in vivo . Since ROur results also provide a molecular explanation for how aRNAs but not other cellular RNAs are specifically recognized by QDE-1. In plants and yeast, it was proposed that RdRPs recognize their specific templates through the recruitment of RdRP by Argonaute-siRNA complexes Although most of the in vitro DdRP products of QDE-1 are DNA/RNA hybrids, some free ssRNA can be generated, as indicated by the generation of some dsRNA from ssDNA. Importantly, we showed that RPA strongly promotes dsRNA production by QDE-1 from ssDNA by preventing the formation of DNA/RNA hybrids. Therefore, RPA may have two roles in this small RNA production process: recruiting QDE-1 to ssDNA and blocking the formation of DNA/RNA hybrids. It has been shown that the human QDE-3 homolog BLM can unwind DNA/RNA hybrids to release free ssRNAs NeurosporaAlthough we use the DNA damage-induced aRNA as a model, the mechanism of aRNA production is most likely shared by quelling, a post-transcriptional silencing mechanism against repetitive transgenes. We showed that, like QDE-1 and QDE-3, RPA is also required for quelling. In addition, QDE-1 is recruited to the transgenic locus upon quelling Neurospora genome. In the case of quelling, which is triggered by the transformation of transgene DNA, a second highly repetitive DNA locus with tandem repeats of transgene is created. Thus, the repetitive nature of the rDNA and the quelled locus is likely to be the cause for aRNA and small RNA production. In Neurospora, the rDNA region is a known site of frequent chromosome breakage The rDNA region is the only highly repetitive DNA locus in the wild-type Arabidopsis, the partially purified RdRP RDR6, which plays an important role in RNAi pathways, has also been shown to have a strong DdRP activity on ssDNA and during dsRNA synthesis, but it cannot distinguish between template RNAs with or without a cap or poly(A) tail in vitro Arabidopsis impairs transcriptional gene silencing at certain loci Neurospora, although it is possible that they may function differently from those in Neurospora. Interestingly, piRNAs from rat testes were found to be associated with rRecQ1 In Neurospora crassa (FGSC4200) was used in this study unless otherwise mentioned. The rpa-3 (NCU01460.3) knock-out strain was obtained from Fungal Genetic Stock Center (FGSC) rpa-1 (NCU03606.3) and dsrpa-2 (NCU07717.3) were created as previously described his-3 locus by electroporation. Strains co-expressing both c-Myc and FLAG tagged proteins were generated by cotransformation of plasmid containing the FLAG tagged gene with plasmid (pBT6) containing benomyl-resistance into the strains expressing the c-Myc-tag protein.A wild type strain of Neurospora, 0.01 M QA (pH 5.8) was added to the liquid culture medium containing 1×Vogel's, 0.1% glucose, and 0.17% arginine. DNA damage was induced by the addition of histidine (100 µg/ml), and samples were harvested 40 h later For the expression of inverted repeats and tagged proteins in qa-2 promoter, creating plasmid qa-3FLAG. Full-length qde-1 or rpa-1 genes were then inserted in frame following the FLAG tag. To create c-Myc-tagged proteins, full-length genes of qde-1, qde-3, or rpa-1 were inserted into a c-Myc tag containing plasmid A FLAG (3×FLAG) tag containing plasmid was a kind gift from Dr. James Chen at University of Texas Southwestern Medical Center. The FLAG tag was subcloned downstream of the Total RNA extraction, enrichment of low molecular weight small RNA, and Northern blot were performed as previously described DA, and RdRP of bacteriophage φ6 were expressed and purified as previously described DA, respectively, each with a carboxy terminal 6-His tag, were introduced into Saccharomyces cerevisiae strain INVSc1 (Invitrogen). The recombinant proteins were expressed at +28°C for 22 h and purified to near homogeneity. Wild-type recombinant φ6 P2 was expressed in Escherichia coli BL21(DE3) strain (Novagen) containing the plasmid pEMG2 Neurospora was partially purified by Ni-NTA matrices (QIAGEN) as previously described Recombinant full-length QDE-1, QDE-1ΔN, QDE-1ΔNSmaI digestion and used as a template for run-off transcription by T7 RNA polymerase. To generate the corresponding ssDNA molecule, pLM659 was used as a template in PCR reactions where one of the primers contained a 5′-biotin. The biotinylated PCR product was immobilized onto Dynabeads MyOne Streptavidin C1 magnetic beads (Invitrogen) according to manufacturer's instructions. The immobilized PCR product was dissolved by treating the DNA briefly with fresh 0.1 M NaOH. The ssDNA was subsequently precipitated and gel purified. For some experiments . For the synthesis of ssRNA of the same sequence, the 176 nt ssDNA was PCR-amplified. The resulting PCR product was purified and used as a template for T7 transcription reaction. The DNA template was then degraded with DNaseI (Promega), and the ssRNA was gel purified. To prepare the 3 kb ssRNA, plasmid pLM659 eriments , the 175RNA polymerase reactions were performed as described Protein extraction, quantification, immunoprecipitation (IP), and Western blot analysis were performed as previously described Neurospora strains were co-transformed with 0.5 µg pBSKal-1 plasmid  gene) and 0.5 µg pBT6 plasmid. The benomyl-resistant colonies were picked and grown on slants. The colors of around 100 transformants from each strain were observed for comparing silencing (quelling) efficiency of the al-1 gene.Figure S1Coomassie-stained SDS-PAGE gels showing the purified truncated (above) and full-length QDE-1 (below). Recombinant QDE-1 proteins expressed in yeast were purified by a Ni-NTA column, a heparin column followed by an ion-exchange column. Protein fractions after the ion-exchange column are shown. The top fractions were pooled and concentrated before use in RNA polymerase assays. Full-length QDE-1 is ∼160 kDa and QDE-1 ΔN is ∼120 kDa in size.(0.20 MB PDF)Click here for additional data file.Figure S2Full-length, recombinant QDE-1 was used in the same RNA polymerase assay as described in , and shown are the ethidium bromide stained native agarose gels. Upper panel: ssRNA template; lower panel: ssDNA template. The activity of the full-length QDE-1 is identical to that of QDE-1 ΔN.(1.26 MB PDF)Click here for additional data file.Figure S332P-labeled ssRNA (left panel) and ssDNA (right panel) templates were used. (B) RNA polymerase reactions using Myc-QDE-1 purified from Neurospora showing that QDE-1 cannot use dsDNA as a template.(A) RNA polymerase reactions showing the association of QDE-1 with ssRNA and ssDNA in the presence and absence of NTP. (0.47 MB PDF)Click here for additional data file.


Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies. The first using oligo-dT primers after polyadenylation of the bacterial RNA, the second using a set of mycobacterial amplification-directed primers both linked to T7 polymerase in vitro run off transcription.The amplification of bacterial RNA is required if complex host-pathogen interactions are to be studied where the recovery of bacterial RNA is limited. Here, using a whole genome M. tuberculosis RNA with unamplified RNA from the same source. In addition, as a direct measure of the effectiveness of bacterial amplification for identifying biologically relevant changes in gene expression, a model M. tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5 ng total RNA . Differential gene expression patterns observed with unamplified RNA in the switch from aerobic to microaerophilic growth were also reflected in the amplified expression profiles using both methods.The reproducibility, sensitivity, and the representational bias introduced by these amplification systems were examined by contrasting expression profiles of the amplified products from inputs of 500, 50 and 5 ng total Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profiling. In contrast to eukaryotic mRNA, techniques to amplify bacterial RNA for use in microarray analyses have not been available until recently, and crucially have not been evaluated for M. tuberculosis in well characterised models in which differential gene expression is well described and validated. Many of the established eukaryotic amplification strategies have been based on the Eberwine method of in vitro transcription with T7 RNA polymerase using oligo-dT primers to selectively amplify eukaryotic mRNA containing poly-A tails [The application of bacterial transcriptomics to complex biological situations is currently hampered by the low amounts of bacterial RNA available after purification from -A tails . However-A tails , this te-A tails . Lately -A tails -7 or ran-A tails ,9 priminMycobacterium tuberculosis microarray. The first priming method involved polyadenylating the bacterial RNA prior to amplification using the Eberwine T7-oligo-dT method. The second priming strategy used a set of mycobacterial amplification-directed primers (with the addition of a T7 promoter sequence) to prime the first strand cDNA synthesis instead of oligo-dT (as in the eukaryotic system) followed by T7 transcript amplification. A minimal set of amplification-directed primers (ADP) was designed to prime all the genes in the combined genomes of M. tuberculosis H37Rv [M. bovis AF2122/97 [et al. [In this study we examine two different methods of bacterial RNA amplification using alternative priming strategies, on the basis that priming efficiencies will have maximum effect on the representation of mRNA populations through possible priming bias. Whole genome representation of the amplified products was measured using a is H37Rv , CDC1551is H37Rv , and M.  [et al. , and whi [et al. -16.M. tuberculosis grown aerobically in vitro) were compared to measure the reproducibility and bias introduced by RNA amplification; (B) RNA extracted from M. tuberculosis cultures under well characterised aerobic and microaerophilic (NRP1) conditions [A number of variables were investigated: size distribution and yield of amplified product, the sensitivity and reproducibility of the amplification systems, the impact of contaminating genomic DNA and eukaryotic RNA, and the mRNA representation bias introduced by amplification. To explore these parameters three experimental designs were required: (A) Amplified and unamplified RNA from the same source  M. tuberculosis microarray against a genomic DNA reference. This strategy allowed the variation and bias introduced by amplification into the gene expression profiles of the same starting RNA to be examined. A second microarray study  was used to identify significantly differentially expressed genes by hybridising amplified RNA from aerobic and microaerophilic (NRP1) culture conditions directly against one another. These significantly differentially expressed genes were then compared to patterns previously determined by microarray analysis from unamplified RNA and from the published literature as a measure of how well biologically relevant transcriptional profiles were represented in the amplified datasets. A third strategy comparing the mycobacterial gene expression profiles derived from mixed RNA populations, where bacterial RNA may be contaminated with eukaryotic host RNA, was used to determine the effect of co-purified eukaryotic RNA on the amplified mycobacterial transcriptional profile. This scenario may be relevant to in vivo models of infection such as experimental murine tissue or human-derived tissue in which bacterial RNA represents a minor component of total RNA.The application of bacterial RNA amplification methods to explore previously inaccessible models of infection by microarray analysis was assessed using three experimental strategies. In the first , unamplified and amplified mycobacterial RNA (extracted from agitated mid-log phase bacilli) were hybridised to a M. tuberculosis amplification-directed primers.The addition of non-template sequence to a primer may adversely modify the stability of a primer-template dimer; we wished therefore to assess the effect of adding a T7 promoter and additional degenerate linker sequence to a specific primer. We measured the specificity and efficacy of the mycobacterial amplification-directed primers by microarray analysis comparing the RNA profile generated after priming with individual 7 nt ADP-specific primer sequences with that primed using the respective 40 nt ADP with additional T7 promoter and degenerate sequence. Using cutoffs of 2 fold signal/background or a signal > 500 to identify successful priming of genes during the RT-labelling reactions, an average of 92–93% of genes  primed with the ADP+T7 sequence were also primed by the ADP-specific 7 nt primer alone. In addition, the average Spearman's rank correlation of genes identified by either cutoff  to be primed by (x5) ADP +/- T7 sequence was 0.89–0.91. The priming specificity of the ADP primers therefore was not significantly altered by the addition of the T7 sequences, consequently the ADP primers were considered suitable to use as M. tuberculosis was amplified using the ADP or oligo-dT priming methods and hybridised to a M. tuberculosis microarray. The performance of these amplification systems was measured by contrasting the yield, minimum input RNA, reproducibility, and how representative the amplified expression profile was compared to unamplified RNA.500, 50 and 5 ng of total RNA extracted from aerated mid-log phase 2-104 fold total RNA depending on priming strategy and input amount. No product however was detected from 5 ng input total RNA using the ADP priming strategy. Both amplification methods therefore (except for ADP at 5 ng) generated sufficient amplified product for microarray analysis. The size distribution of the aRNA populations differed in modal product size which varied with amplification method and input RNA amount  against all others (including unamplified); secondly, by using an arbitrary 2.5 fold cut-off to identify gross differences in the signal ratio of amplified to unamplified; and thirdly, by comparing the number of genes below a signal threshold to identify the potential loss of information from RNA amplification. A correlation matrix comparing the ranked mean signal of each gene from the amplified products for each amplification strategy with input of 500, 50 and 5 ng of RNA with unamplified RNA was performed  compared to unamplified RNA. Using the oligo-dT strategy 907, 951 and 800 genes were identified to be 2.5 fold over or under represented after amplification with starting amounts of 500, 50 and 5 ng respectively.A third parameter was considered to assess the potential problem that genes may be poorly amplified by the respective amplification system to an extent that the signals were not detected by microarray analysis resulting in the loss of data. The number of genes under a 2 fold median signal/background ratio in the RNA channel was compared between amplified and unamplified samples. The smallest number of 'absent' genes below this detection threshold was 605 and 592 (500 and 50 ng of input RNA respectively) using the oligo-dT amplification method. However the number of undetected genes nearly trebled with a further reduction of input RNA to 5 ng using the same amplification strategy, increasing the number of genes below the background threshold to 1606. A greater number of genes, 2232 and 2296, were not detected using the ADP method at 500 and 50 ng of input respectively. A comparison of genes not detected by microarray analysis from 500 and 50 ng input RNA for each method together with the unamplified RNA controls is depicted in Figure M. tuberculosis RNA extracted under well-defined aerobic and microaerophilic growth conditions was amplified and compared. Significant differences in gene expression identified from this model system were evaluated against a comparison of unamplified RNA and published data.Thus, both amplification systems were applicable to microarray analysis in both yield and reproducibility; both methods were also representative of the unamplified RNA population to a large extent (with correlation coefficients of approximately 0.8) although differences in RNA profile could be detected that may detrimentally affect direct comparisons of amplified to unamplified RNA. Although useful for investigating the accuracy of these bacterial amplification systems, the above experimental design examining unamplified and amplified RNA from the same source did not allow for a comparison of amplification methods in a microarray experiment designed to extract meaningful biological information. Therefore M. tuberculosis H37Rv were determined in aerobic growth compared to growth under conditions of limited oxygen . These microaerophilic conditions were chosen because the experimental procedures have been previously defined [The changes in transcriptome profile of  defined  and a su defined ,18. In o defined ,18.M. tuberculosis in microaerophilic compared to aerobic growth conditions. Furthermore the number of significantly induced genes declined as the input RNA decreased from 500 to 50 to 5 ng using this amplification method. This pattern is further illustrated in Figure 91, 61 and 20 genes were identified as significantly induced in NRP1 compared to aerobic growth using the oligo-dT amplification method using 500, 50 and 5 ng of RNA in the amplifications. Whist 24 and 42 genes were significantly induced in the comparison using ADP amplification from 500 ng and 50 ng input RNA respectively. These gene lists were compared with a gene list of 100 significantly induced genes derived via the same statistical measures in an aerobic vs. NRP1 comparison using unamplified RNA. Of the 100 genes identified as induced in the unamplified comparison 61 overlapped with the significant genes derived from the 500 ng oligo-dT analysis, 40 from 50 ng and 13 from 5 ng oligo-dT amplification. 11 genes overlapped with the significantly induced gene list from the ADP 500 ng microarray analysis, and 21 from ADP amplification of 50 ng input RNA. The oligo-dT amplification strategy therefore correlated most closely with the changes in gene expression identified using unamplified RNA extracted from -41 using unamplified RNA, 3.5 × 10-32, 9.6 × 10-24, and 8.2 × 10-7 using the oligo-dT amplification method , and 0.15, 1.9 × 10-7 using the ADP amplification strategy. Similarly the devR (dosR) regulon previously identified to be induced by hypoxia [-71 using unamplified RNA, 5.2 × 10-47, 1.0 × 10-28 and 3.4 × 10-6 in the oligo-dT comparison , and 0.26, 1.2 × 10-9 in the ADP amplified comparisons. The hypergeometric probabilities for the ADP comparisons were significant only at an input of 50 ng total RNA; this may reflect suboptimal performance of the reverse transcription and a need to optimise the primer:template ratio. However, biologically significant patterns of gene expression may be identified by microarray analysis of amplified RNA from as low as 5 ng total RNA input.The biological patterns of gene expression associated with the shift from aerobic to microaerophilic growth could be readily identified using amplified RNA. The hypergeometric function, that statistically tests for the probability that functional categories of genes are significantly enriched, was used to determine whether previously defined changes in gene expression pattern were identifiable in the gene lists generated in the aerobic vs. NRP1 comparisons using amplified RNA. Genes previously observed to be induced during non-proliferating conditions  were enr hypoxia  was alsoM. tuberculosis RNA as input into the two amplification systems, analogous to RNA extracted from in vitro conditions or from infection models where a differential lysis method of extraction . The impact therefore of contaminating levels of eukaryotic RNA after amplification (using either method) and hybridisation to a M. tuberculosis microarray was minimal. This correlates with the findings of Lawson and Johnston [The studies above used purified total traction -22) is aJohnston  using LiM. tuberculosis total RNA was successfully amplified and analysed by microarray analysis from between 5 to 500 ng of total RNA. The oligo-dT method, based on the polyadenylation of RNA followed by oligo-dT primed amplification, consistently gave the greatest yield of aRNA. The ADP method, using a subset of primers designed to initiate cDNA synthesis within the terminal region of all coding sequences within the M. tuberculosis H37Rv, CDC1551 and M. bovis AF2122/97 genomes, generated similar yields with 500 or 50 ng input RNA but failed to provide any significant product from 5 ng of input RNA. This may reflect suboptimal performance of the reverse transcription and the need to optimise primer:RNA ratio since lowering the primer input for a fixed amount of RNA affected the size distribution of the amplified product using this amplification system (data not shown). This may also account for the greater biological significance of comparisons from an RNA input of 50 compared to 500 ng using the ADP method. The observed differences in modal size between the ADP (~750 bp) and oligo-dT (~1600 bp) methods can be explained by the initial priming event during cDNA synthesis since all other aspects of these techniques were the same. ADP primers were designed to initiate cDNA synthesis within 500 nucleotides of the 3' of the coding sequences of each gene, however primers could bind elsewhere in the ORF. Thus ADP priming will initiate cDNA synthesis at multiple points within the message, in contrast oligo-dT priming will only occur within the terminal polyadenylated tails added to the 3' end of the polycistronic bacterial message (or indeed intragenic polyA sequences); this pattern was also observed by Rachman et al. [n et al. . TherefoThe representative nature of the amplified products compared to unamplified RNA was measured by Spearman's rank correlation, and by applying fold change and signal/background thresholds. Correlation coefficients ranging from 0.79 to 0.84 were measured between amplified products from both amplification systems and unamplified RNA. This compares to other methods of amplification with correlation coefficients of amplified to unamplified of 0.44–0.75 for inputs of 10–5000 ng bacterial RNA ; or 0.73The potential loss of expression data as a consequence of poor or inefficient gene specific amplification was investigated by comparing the number of genes flagged as absent in the microarray analyses. The number of undetected genes was higher after amplification using the ADP system compared to unamplified RNA, furthermore the number of predicted ADP primer-binding events did not appear to be a significant factor in the loss of detectable genes from the ADP amplified analysis. However lower signal intensities from a subset of genes using the ADP amplification method may lead to an increased number of aberrant expression ratios, and therefore decreased levels of significance when applied as part of down-stream data analysis. Correlation coefficients 0.90 – 0.98) between products amplified from different input amounts using the same amplification method demonstrated that the gene expression pattern of amplified RNA was similar, independent of input amount. This may be an important factor as variation in RNA input amount might be unavoidable in some experimental settings. The number of genes below the detection threshold however increased as RNA input into the amplification reactions decreased. This pattern of reduced sensitivity with decreasing input RNA was also observed using Whole Community RNA Amplification [0 – 0.98 M. tuberculosis microarray. This should allow for the successful transcriptome analysis of bacterial RNA extracted where the co-purification of at least equal quantities of contaminating eukaryotic RNA is unavoidable.Little or no amplification products were detected when amplifying RNase treated samples, illustrating the minimal impact of contaminating genomic DNA on RNA amplification from the samples used in this study; however heavily contaminated samples may present difficulties and DNase treatment of RNA before amplification is essential. Additionally, product was amplified from 250 ng eukaryotic RNA using both amplification methods; in mixed RNA population amplifications however there was little or no effect of the contaminating eukaryotic RNA on bacterial transcriptional patterns measured using a et al. [et al. [The amplification systems detailed in this study generate anti-sense aRNA, which may present difficulties if this product is to be labelled during cDNA synthesis of the amplified product for hybridisation to oligonucleotide microarrays . Rachman et al.  and Laws [et al.  have recM. tuberculosis RNA; the amplification of mycobacterial ribosomal RNA that may have overwhelmed the amplification reactions and subsequent hybridisations was not problematic. Whether this was due to the high tertiary structure of ribosomal RNA limiting primer binding, 3' modifications limiting polyA tailing or an excess of reagents is unknown. The major advantage of being able to successfully analyse gene expression patterns from amplified total RNA is the elimination of the requirement for mRNA purification from total RNA extractions.The bacterial amplification systems described here were tested on total From this work we offer the following general recommendations for the amplification and microarray analysis of bacterial RNA: (A) remove contaminating DNA as it will likely be amplified alongside the bacterial RNA population; (B) remove contaminating eukaryotic RNA before amplification or assess the impact of co-purified eukaryotic RNA on amplified bacterial gene expression patterns; (C) amplify from similar input amounts of RNA to reduce variation between replicate samples; (D) amplify and hybridise samples from a single experiment together where possible; and crucially (E) do not compare amplified directly to unamplified RNA, or to RNA amplified using an alternative method.in vitro transcript amplification following 2 methods of cDNA priming and define the limitations of this topical area of bacterial RNA amplification; the successful use of which will enable researchers to study previously intractable host-pathogen interactions where bacterial RNA is limiting.The recognition of biologically relevant pathways in gene expression profiles derived from amplified RNA in the model system tested here confirms for the first time that the amplification of bacterial RNA may be used successfully in bacterial transcriptomics. RT-PCR validation will still be required for single gene inferences of biology, ideally from total RNA before amplification. However the advantages of microarray analyses, using changing patterns of transcription  to interpret biologically significant scenarios, are still applicable using amplified RNA. This should facilitate the study of complex host-pathogen interactions that previously could not be investigated because of low bacterial numbers, an exhaustive number of timepoints/conditions, or low RNA extraction yields. In summary, we describe the RNA populations generated by Mycobacterium tuberculosis H37Rv was grown as agitated cultures (370 rpm) to mid-log phase at 37°C in Dubos liquid medium, supplemented with Dubos medium albumin. M. tuberculosis microaerophilic (NRP1) cultures were set up and cultured in a stirred model for 72 h according to Wayne and Hayes [et al. [nd Hayes . Mycobac [et al. ; RNA waset al. [M. tuberculosis H37Rv [M. bovis AF2122/97 [M. tuberculosis H37Rv RNA was labelled for microarray hybridisation using reverse transcriptase with 2 μM ADP primer +/- the 5' anchor, T7 polymerase binding sequence, and random four nucleotide sequence. The products from five ADP (+/- T7 sequences) primed RT-labelling reactions were hybridised independently against genomic DNA to the M. tuberculosis microarray (as described below), and the signal from the RNA-derived products compared to measure the effect of the additional T7 sequence on ADP primer specificity.A minimal set of amplification-directed primers (ADP) were designed in an iterative fashion using an algorithm similar to that described by Talaat et al.  to bind is H37Rv , CDC1551is H37Rv , and M. M. tb amplification-directed primers (ADP) were used in the place of oligo-dT in the MessageAmp II eukaryotic amplification system (Ambion); the second priming strategy included an initial polyadenylation step (with E. coli polyA polymerase at 37°C for 15 minutes) followed by oligo-dT based amplification . Amplification reactions were conducted according to manufacturers instructions, with inputs of 500 ng, 50 ng and 5 ng total mycobacterial RNA. Single rounds of amplification were performed, with an IVT reaction of 16 hours at 37°C. Control reactions containing 500 ng M. tb H37Rv genomic DNA, 500 ng RNase-treated M. tb total RNA or water were also performed using both amplification systems. Amplifications were repeated in duplicate on two or more separate occasions. To determine the impact of contaminating eukaryotic RNA, 250 ng M. tb H37Rv total RNA +/- 250 ng eukaryotic total RNA, and 250 ng eukaryotic total RNA alone was amplified in triplicate using both amplification methods. The yield and size distribution of all amplified products was assessed spectrophotometrically at OD260 and using the Agilent 2100 Bioanalyser (Agilent Technologies).50 μM M. tuberculosis whole genome microarray consisting of 4410 gene specific PCR products  to the M. tuberculosis H37Rv [M. bovis AF2122/97 [M. tuberculosis H37Rv RNA (aerated mid-log phase in vitro cultures) against 2 μg Cy3-labelled M. tuberculosis H37Rv genomic DNA (provided by Colorado State University). In the second comparison  5 μg cDNA derived from amplified aerobic RNA was labelled with Cy3 and hybridised against 5 μg cDNA derived from amplified NRP1 (microaerophilic) RNA labelled with Cy5. The hybridised slides were scanned sequentially at 532 nm and 635 nm corresponding to Cy3 and Cy5 excitation maxima using the Affymetrix 428™ Array Scanner (MWG). Comparative spot intensities from the images were calculated using Imagene 5.5 (BioDiscovery), and imported into GeneSpring GX™ 7.2 (Agilent Technologies) for further analysis. The array data were normalised to the 50th percentile of all genes detected to be present on the array. In the first microarray experiment (A) the expression of all M. tuberculosis H37Rv genes were analysed . Spearman's rank correlations and over/under-represented genes were generated from combining replicate hybridisations of each condition (with a minimum of 2 and maximum of 6 replicates/condition). In the second (B) aerobic vs. NRP1 experiment two independent amplifications were conducted and hybridised in duplicate. These 4 hybridisations were combined together before significantly differentially regulated genes were identified between aerobic and microaerophilic (NRP1) conditions using a t-test p-value < 0.05 with Benjamini and Hochberg multiple testing correction and a > 2.5 fold cutoff. A minimum of 5 replicate hybridisations were conducted with unamplified aerobic and NRP1 RNA before defining statistically significant genes as above. The hypergeometric distribution was used to determine if previously published functional categories of genes were significantly enriched in the amplified comparisons [A is H37Rv , and M. F2122/97 , array aF2122/97  with, inparisons . Fully aparisons  and alsoin vitro transcription; aRNA, Amplified RNA.ADP, Amplification-Directed Primers; NRP1, Non-Replicating Persistence Stage 1; IVT, SJW designed the study and carried out the amplifications and amplified RNA microarray analyses and also drafted the manuscript. KL designed the amplification-directed primer set, helped analyse the microarray datasets and draft the manuscript. CS extracted the RNA and performed the unamplified RNA microarray analysis. PDB conceived of the study and helped draft the manuscript. All authors read and approved the final manuscript.47 amplification-directed primer sequences. The nucleotide sequences of the amplification-directed primers designed and utilised in this study.Click here for file


Robustness, maintaining a constant phenotype despite perturbations, is a fundamental property of biological systems that is incorporated at various levels of biological complexity. Although robustness has been frequently observed in nature, its evolutionary origin remains unknown. Current hypotheses suggest that robustness originated as a direct consequence of natural selection, as an intrinsic property of adaptations, or as a congruent correlate of environment robustness. To elucidate the evolutionary origins of robustness, a convenient computational package is strongly needed. under the GNU/GPL license. We present an overview of EvoRSR package and illustrate its features with the miRNA gene cel-mir-357.In this study, we developed the open-source integrated system EvoRSR  to explore the evolution of robustness based on biologically important landscapes induced by RNA folding. EvoRSR is object-oriented, modular, and freely available at EvoRSR is a novel and flexible package for exploring the evolution of robustness. Accordingly, EvoRSR can be used for future studies to investigate the evolution and origin of robustness and to address other common questions about robustness. While the current EvoRSR environment is a versatile analysis framework, future versions can include features to enhance evolutionary studies of robustness. Robustness is a fundamental and ubiquitous phenomenon in biological systems, in which phenotypes are resistant to change in the presence of various perturbations. When these perturbations are inherited, such as genetic mutations, the phenomenon is known as genetic robustness. Alternatively, when the perturbations are due to environmental factors, the phenomenon is called environmental robustness . Both tyAddressing these questions requires a convenient computational package that will fully elucidate the evolutionary origins of robustness. A good example to study for clarifying the origins of robustness is RNA folding from sequences into secondary structures. RNA folding provides a convenient biophysical model of a genotype-phenotype map that has been used in studies for robustness, evolvability, and epistasis. In such studies, RNA folding can be precisely defined and statistically measured, revealing simultaneous and non-independent effects of natural selection ,9. These under the GNU/GPL license. This open-source package inspects the evolution and origin of robustness through sampling genotype (sequence) space at each Hamming distance from the WT sequence. Here, we describe the EvoRSR package and analyze the miRNA gene cel-mir-357 to illustrate how EvoRSR works.Given a quantitative measure of structural robustness ,18,19, wFigure The EvoRSR package is a free package written in C, which runs in a command-line mode within a Linux/Unix environment. The Vienna RNA package  is requil is defined asFormally, the neutrality η of an RNA sequence with length d is the base-pair distance between the secondary structures of the WT sequence and its mutant, averaged over all 3 × l one-mutant neighbors. d is calculated by RNADISTANCE in Vienna RNA package [dG, is quantitatively measures the thermodynamic stability  of a WT RNA sequence [dG is calculated as the minimum free-energy of secondary structure obtained by RNAFOLD in Vienna RNA package [Evoneu is applied to calculate the η s and dGs of the sequences in a Fasta file  is replaced by the general multi-structure distance between the ensemble of secondary structures of the WT sequence and its mutant [dG is replaced by the ensemble free-energy.Because RNA molecules may function in dynamical, structural reconfigurations ,23, an Rs mutant , and theS, which can be divided into subsets Si, i = 1, 2, ..., l that represent the set of sampling sequences within a Hamming distance of i from the WT sequence. All the subsets have an identical size, .For each WT RNA sequence, we employ a Monte Carlo method to sample sequences in the genotype space at each Hamming distance from the WT RNA sequence. The set of total sampling sequences is denoted by P(t|h) is the conditional probability that given two sequences Hamming distance h from each other, the two configurations have either a base-pair distance t or a free-energy difference t. The density surface describes how the distributions of free-energy values and configuration differences change along the Hamming distance from the WT sequence. Furthermore, the density surface condenses statistical aspects of the correlation between sequences and structures and provides a tool to derive and calculate local and global properties of sequence-structure relations.As a generic tool for the study statistical properties of landscapes, we propose the use of a two-dimensional probability density surface ,26. A deLandscape can be characterized statistically by autocorrelation functions ,28, whicd2⟩ is the mean squared distance sampled over the entire sequence space, and h) are approximated by an exponential fit to calculate a correlation length ℓ for secondary structures in sequence space:⟨l [Evoautocf  to explore the evolution of robustness based on biologically important landscapes induced by RNA folding. EvoRSR is object-oriented, modular, and freely available at Project name: EvoRSR Project home page: Operating system(s): Linux, UNIX (no GUI)Programming language: C++ and PerlOther requirements: Vienna RNA packageLicense: GNU/GPL licenseRestrictions to use by non-academics: NoneThe authors declare that they have no competing interests.WS and MN wrote the programs, analyzed the results. WS drafted the manuscript. XB and ZZ helped in analysis and discussion, gave useful comments. SW and XB guided the project. All authors read and approved the final manuscript.cel-mir-357The results of . The C. elegans microRNA (miRNA) mir-357 (cel-mir-357) is analyzed as example to illustrate how EvoRSR can be helpful for studying the evolution of robustness.Click here for file


The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity.We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants.Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions. As the site of oxygenic photosynthesis, chloroplasts are the most in-depth studied plant organelles. The recognition that they carry their own DNA  led, in rpo genes encoding subunits of a homolog of the eubacterial RNA polymerase, termed plastid-encoded RNA polymerase (PEP). Surprisingly, in contrast to its eubacterial ancestors, this RNA polymerase does not transcribe the chloroplast genome in higher plants alone , wheed sites . In cyaned sites ,33. Althed sites . Thus, croplasts . Those troplasts  and are roplasts ,14,36,37roplasts ,38. Besiroplasts -41. In cChlamydomonas reinhardtii. Here, the abundance of the chloroplast PPR protein MCA1 has been demonstrated to limit the amount of the petA mRNA as well as the amount of the encoded protein, cytochrome f . For exchrome f ,46. A vachrome f -49. Nonechrome f ,51. In cd plants -55. TakeAlthough positive selection of a complex RNA metabolism of chloroplasts remains a potent possibility for explaining its having come about, we think that there is at least one powerful alternative explanation. In short, this hypothesis puts forth the proposition that complexity in organellar gene expression can be a selectively neutral process, fostered by different mutation rates and a different strength of genetic drift between the organellar and nuclear genomes of plants.Buchnera, an endosymbiont of aphids, accumulate mildly deleterious mutations . Thus, in vitro and are by definition ribozymes. Chloroplast group II introns, however, have lost their ability to excise without external help. This might also point to the detrimental effects of Muller's ratchet being active in chloroplasts. For splicing, nuclear encoded factors are required . Intereiewed in -11. It sBesides coding regions and introns, Muller's ratchet should also lead to the weakening or even destruction of expression signals in the 5' and 3' UTR of mRNAs. Sequence elements in the UTR regions are required for RNA stability, but (particularly in the 5' UTR) also for translation initiation. Indeed, the canonical Shine Dalgarno sequence in front of cyanobacterial genes is rarely found in the typical position in front of chloroplast open reading frames, if it is found at all . ChlMany point mutations can be cured on the RNA level by nuclear factors, i.e. highly specific RNA binding proteins. In contrast, processes depending on DNA as a template will require other means to compensate for deleterious point mutations. These DNA-bound processes are chiefly replication and transcription. Only some features of the replication machinery in chloroplasts are known  but, clArabidopsis thaliana, grapevine (Vitis vinifera), rice (Oryza sativa) and moss Physcomitrella patens genome is six; nine were found in poplar (Populus trichocarpa), but only one is present in the sequenced green algal genomes of C. reinhardtii, Ostreococcus tauri and Ostreococcus lucimarinus  revealed a CRT-motif (CAT or CGT) at position -8 to -6, which was proven to be critical for transcription  or commUGM conceived the study. SAR searched the available data for transgenomic suppressors. All authors discussed the results, wrote and approved the final manuscript.


Self-rated health (SRH) is a robust predictor of subsequent health outcome, independent of objective health measures and life-style-related health risk factors. However, the determinants of SRH are as yet largely unknown. In accordance with the prevailing stress theory, we hypothesized that SRH is associated with personal coping resources, psychological strain, life-style variables, and endocrine variables.A total of 106 healthy women, 22–59 years of age, were followed for up to 3 years with annual blood sampling  and written questionnaires in which information on SRH, psychological strain, coping resources, socio-economic and life-style variables was sought.P < 0.0001), fitness (P = 0.0002), and sense of coherence (P = 0.0006) were independently associated with SRH, together explaining 74% of the SRH variance.In bivariate, screening logistic regression analyses, intended to find candidate variables for a final analysis model, all coping resource variables  were significantly related to SRH, and so were two psychological strain variables , one life-style variable (fitness), but none of the endocrine variables. In the final multivariate analysis model, including all candidate variables, only vital exhaustion (Some elements of the hypothesis, i.e. the effects of coping resources, psychological strain, and life-style variables on SRH, were supported by the results, while others, i.e. effects of endocrine measures on SRH, were not, indicating a possible gender difference. Although attenuated, the association between SRH and health remains, even when objective health measures and known health risk factors are controlled for .It has been suggested that stress theory-based psychobiological mechanisms may explain part of the predictive validity of SRH ,10. SRH The hypothalamic-pituitary-adrenal (HPA) axis is one of the primary effectors of the stress response systems. Chronic stress has been associated with increased levels of serum prolactin, decreased levels of serum testosterone, and with increased or decreased levels of serum cortisol, the latter having been suggested to occur following long-term exposure to chronic stressors .Few studies investigating the suggested association between SRH and stress theory-based mechanisms have included concurrent measures of activity in the endocrine stress response, psychological strain, and personal coping resources ,14,23,24The aim of the present study was therefore to investigate associations between stress theory-based endocrine markers, psychological strain, personal coping resource variables, and SRH in a sample of healthy adult women. We hypothesized that SRH would be positively and independently associated with personal coping resources in terms of sense of coherence, mastery, and self-esteem, and with levels of testosterone, and negatively associated with psychological strain in terms of psychological symptoms, sleep disturbances, and vital exhaustion, and with levels of cortisol and prolactin.The present study is part of a longitudinal project focusing on globalization of work, with a study population recruited as subjects or partners of subjects with planned assignments abroad, or as matched non-moving controls . The stuParticipants responded to a written questionnaire and had blood samples drawn at base-line and at 1-year intervals during the follow-up period, for a maximum of 3 years. The number of follow-up measurements performed varied, depending on the length of assignment abroad for the participants in the moving group, with the non-moving group followed for a corresponding period of time. The participation rate throughout the study was 81.1%. Reasons for non-participation at base-line and drop-out during follow-up are given in The written questionnaire included questions concerning SRH, personal coping resources, psychological strain, life-style factors, age, educational level, employment status, and medication. SRH was measured using a one-item global question phrased ‘How would say your general health has been during the past year?’ Possible responses ranged from ‘bad’ (=1) to ‘excellent’ (=5). Personal coping resources were assessed using the Pearlin 7-item mastery scale, which measures generalized beliefs about control , the RosPsychological strain was measured using a 12-item version of the general health questionnaire (GHQ) scale . The 21-Leisure time exercise was classified on a scale ranging from ‘never’ (=1), to ‘regularly, more than once a week’ (=4). Self-rated fitness was classified on a scale ranging from ‘very poor’ (=1), to ‘very good’ (=5). Smoking habits were classified as currently being a smoker (=1) or a non-smoker (=0). Frequency of any type of alcohol intake in order to relax after work was classified on a scale ranging from ‘less than once a month’ (=1), to ‘daily’ (=4). For this report, educational level was classified as ‘compulsory or vocational school only’ (=1), ‘college’ (=2), ‘university level’ (=3), or ‘other’ (=4); employment status was classified as working ‘full time’ (=3), ‘part time’ (=2), or ‘no gainful work’ (=1); and medication was classified as ‘no medication’ (=0), ‘less than daily but at least once a week’ (=1), or ‘daily’ (=2).Venous blood samples were drawn between 8 a.m. and 10 a.m., following an overnight fast. They were centrifuged, and frozen at −20°C for later analysis at the Department of Clinical Chemistry, Karolinska University Hospital, which has an approved quality control/quality assurance (QC/QA) programme. Serum levels of prolactin and cortisol were analysed using time-resolved fluorescence immunoassay kits . Serum testosterone levels were analysed using a RIA kit from Diagnostic Products Co . The coefficients of variation were 6.2% for prolactin, 8.2% for cortisol, and 12.8% for testosterone.An ordinal time variable was created to identify when blood and questionnaire data were collected . Participants were categorized as pregnant, breast-feeding, or non-pregnant/non-breast-feeding, on each measurement occasion, based on verbal information in connection with blood sampling, on questionnaire data concerning number of children presently living in the household, and on levels of oestrogen and prolactin in blood sampled. The study was approved by the Research Ethics Committee at the Karolinska Institute, Stockholm (KI D No 91:28).Statistical analyses were performed using SPSS 13.0  and SAS P-value is based. As a consequence, Wald's chi-square estimate may be used to rank the impact, or importance, of the independent variables, and also to assess the contribution of the various levels to the total variance in multilevel analyses.In order to make full use of SRH as a five-level ordinal variable, multivariate ordinal logistic regression was used, providing pooled odds ratios (OR) across the five SRH levels for each independent variable, confidence intervals, and Wald's chi-square estimate . The lasChange in SRH over time was non-isotonic, i.e. did not increase or decrease consistently across time, and the number of measurement occasions varied between subjects. To make full use of available data a cross-sectional analytical approach was used, based on a concatenated data arrangement, which means that data from each measurement occasion for a subject constituted an observation, or ‘data line’. Altogether 340 observations were produced by the 106 participating women, with their, on average, 3.2 measurement occasions.A potential problem with this analytical approach is data dependence, since concatenated data are treated as if all measurements are independent, although up to four data lines may refer to the same subject. On the other hand, the measurement occasions were one year apart, diminishing the degree of dependence. Four methods were employed to check the degree of data dependence. First, an ordinal logistic regression-based multilevel analysis was performed on concatenated data, showing a very low degree of dependence. Furthermore, a cross-sectional analysis of base-line data only, an analysis based on mean values across time for each variable, and an analysis based on individual regression coefficients across time all showed similar results as those based on concatenated data, the only difference being that the concatenated data-based analysis had by far the highest statistical power. The last-mentioned was therefore used in this study.Since a large number of potential independent variables, or SRH determinants, were involved, these were grouped into endocrine variables , life-style variables , personal coping resource variables , and psychological strain variables .The analyses were performed in two steps. In the first, screening step candidate variables for further analysis were identified in each independent variable group, through multivariate ordinal logistic regression analyses, with SRH entered as outcome (dependent) variable, and the variables in each group entered as independent variables, one group at a time. Age, smoking and alcohol habits, pregnancy or breast-feeding, and oestrogen levels were entered as covariates, to adjust for their potential influence on outcome. In the second step, the significant candidate variables identified in the screening analyses were, together with the covariates, entered as independent variables in a final multivariate ordinal regression analysis model with backwards elimination of non-significant variables.Post hoc power analyses based on the association of vital exhaustion and sense of coherence, on the one hand, and SRH on the other in this study, showed a beta of >90%, given the size of the study population, and an alpha of 0.05. For the effects of cortisol on SRH the statistical power was just short of 80%, while the power analyses of testosterone and prolactin on SRH indicated low power due to small effect size.P < 0.05 in the screening analyses and at P < 0.01 in the final analyses model, to account for multiple testing. Confidence intervals were computed accordingly.All tests were two-tailed. Significance levels were set at Study-population characteristics are presented in Table I. Mean age was 37 years, range 22–59. At base-line, more than 80% rated their health as excellent or good. More than half had university education. A majority of the participants were married or co-habitants, in full-time or part-time work, and had children aged less than 5 years living in their household. Almost half of the participants reported regular exercise at least once a week, and a majority described their fitness as average. Less than one-fifth of the participants were smokers. The vast majority reported alcohol intake for relaxation purpose less than once a month. A total of 6% were on regular medication.Data concerning endocrine measures, personal coping resources, and psychological strain variables are presented in Results of the screening analyses are shown in P < 0.01 level. SRH was inversely associated with vital exhaustion and positively associated with fitness and sense of coherence. No other variable remained significantly associated with SRH. The final model explained 73.7% of the SRH variance.Based on the results of the screening analyses mastery, self-esteem, sense of coherence, vital exhaustion, sleep disturbances, and fitness were entered as independent variables into the final ordinal logistic regression analysis model with SRH as dependent variable (outcome) and age, smoking, alcohol in the evening in order to relax, on-going pregnancy/breast-feeding, and oestrogen entered as covariates. The results are presented in The independent effects of vital exhaustion, fitness, and sense of coherence on SRH are visualized in There was a strong association between vital exhaustion, fitness, and sense of coherence on the one hand, and SRH on the other, with the final regression model explaining almost 74% of the observed SRH variance. No statistically significant associations were observed between endocrine variables and SRH. To our knowledge, this is the first study investigating associations between endocrine as well as psychological stress theory-based variables and SRH, in healthy middle-aged women.The study takes its departure point in the hypothesis that SRH may be viewed as a potential risk indicator of sustained responses to stressor exposure, and that stress theory-based mechanisms explain part of the predictive validity of SRH in relation to future health. An important question concerns the validity of independent variables investigated in relation to the research question posed. Face validity of vital exhaustion and sense of coherence may, from a stress theory-based perspective, be considered good. Vital exhaustion, a state of exhaustion thought to occur in response to prolonged psychological stress, is conceptually similar to burn-out, and empirically close correlations with various burn-out measures have been reported . The SOCRegarding criterion validity, vital exhaustion has, in longitudinal studies, independent of known risk factors for cardiovascular disease and severity of concurrent disease, been associated with increased risk of cardiovascular disease and with all-cause mortality ,37,38. SMean and median levels of vital exhaustion in the present study corresponded to levels previously reported in healthy study populations . Mean leThere was a strong inverse association between psychological strain, in terms of vital exhaustion, and SRH. Results are in line with observations concerning healthy adult men based on data from the same original study population  and in lIn line with previous research there was a strong positive effect of personal coping resources, in terms of sense of coherence, on SRH, ,44. ResuPost hoc power analyses indicated a low statistical power in the analyses of testosterone and prolactin on the one hand, and SRH on the other, not due to a too-small study population but to a too-small effect size, indicating that the variables in question may be associated, but that the direct effect of the independent variable on the dependent variable is weak. Applied to the present study, results thus suggest that testosterone and prolactin may be associated with SRH, but that the associations perhaps are mediated by other variables, attenuating the direct relationship. Associations between prolactin, testosterone, and SRH have previously been reported in healthy adult men, based on data from the same original study (. Bivariate associations between prolactin and SRH were observed in a recent study of adult women (Importantly there were no statistically significant associations between endocrine variables and SRH. al study , one of lt women . The preThere were no significant associations observed between cortisol and SRH, which is in line with results from two previous studies based on male study populations ,24. HoweAssociations between life-style factors and SRH are well established. In the present study, perceived fitness was a strong determinant of SRH. Results thus underline the importance of including life-style variables in regression models aimed at understanding the role of potential predictors of SRH. Contrary to expectations, smoking was positively associated with SRH.Limitations of the study concern questionnaire data on some of the life-style-related variables. For smoking and alcohol consumption, data were somewhat crudely measured. Similarly, endocrine data were based on sampling performed once on each measurement occasion; for increased precision, repeated blood sampling at each measurement point in time would have been desirable. Furthermore, associations between SRH and salivary, but not serum, cortisol levels have been reported , raising. A further strength of the study concerns the use of ordinal regression modelling (The main strengths of the study include that the study population consisted of healthy women with low levels of health risk behaviours. Associations observed between independent variables and SRH can thereby not be explained in terms of effects of concurrent disease or disabilityodelling ,45. A maAnalyses were cross-sectional, and causal inferences can therefore not be made. Repeated studies, based on larger study populations, and repeated blood sampling, with results reported separately for women and men, are required, to test the reliability of the present results. Finally, the present study was focused on investigating associations between stress theory-based psychobiological variables and SRH. Future studies need to determine if, and to what extent, observed associations contribute to the predictive validity of SRH in relation to future health.Results supported the hypothesis that personal coping resources and psychological strain variables affect SRH but gave no evidence of associations between endocrine variables and SRH in this sample of healthy adult women.


MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles.Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP) or microRNA-specific real-time quantitative PCR (miQPCR). Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays.MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven. MicroRNAs (miRNAs) are evolutionary conserved, small, non-coding RNAs that post-transcriptionally control the expression of protein coding genes ,2 in divTo our knowledge, RNA integrity and its effect on miRNA detection has not been studied in snap-frozen specimens. We therefore addressed the question to what extent total RNA degradation affects miRNA profiles. Our data show that total RNA degradation in defrosted tissues significantly affects miRNA integrity. The lower the quality of total RNA, the higher the proportion of miRNAs that show aberrant signal intensities in microarray and qPCR based approaches. Importantly, we could not identify any systematic effect on the degradation of specific miRNAs that would allow for appropriate correction. Based on these findings, we concluded that reliable miRNA expression profiles using microarrays and qPCR are only achieved if total RNA with RNA Integrity Number (RIN) equal to or above seven is used.RNA can be readily damaged by heat, UV, acid or base catalyzed hydrolysis and by enzymatic degradation. However, individually these approaches do not replicate the diverse insults that occur during the extraction of total RNA from frozen tissues. To mimic total RNA degradation in snap-frozen livers and duodenums from mice, tissues were stored in a -80°C freezer and then transferred to dry ice. Each frozen tissue was sliced into five identical pieces which then were transferred into eppendorf tubes. At time point zero (T0), all samples were placed on ice and total RNA was extracted immediately from (T0) and at later time points . RNA integrities were assessed using the Agilent Bioanalyzer 2100 which calculates RIN values of assayed RNAs  that accurately and sensitively monitors the genome-wide expression of known mature miRNAs and that discriminates between closely related miRNA family members ,21. To aversus low RINs  and analyzed it by the miQPCR method (manuscript in preparation). The abundance of seven liver-expressed miRNAs  and of RNU6 was analyzed by using SYBR Green and the crossing threshold value  samples were moved into ice and at different time points 1 ml of Trizol (Invitrogen) was added to the sample. Tissues were immediately disrupted by using mechanical shearing . Total RNA containing small RNA was extracted from the homogenate as previously described ,21. RNA 32 by using PNK . Signal intensities were recorded by using phosphoimager (Toshiba) and normalized by using non-saturated images of ethidium bromide stained gel  taken before transfer to the membrane. Signal intensities were calculated using ImageJ  and run on a ABI 7500 instrument following standard 7500 mode  with dissociation step (ramping from 60°C to 95°C) for monitoring melting curve of the PCR products. After the run threshold cycles  repository and are accessible under accession number GSE18788 and GSE19104.DI, VB, MUM and MC declare no competing financial interests.DI performed RNA extraction and microarray experiment; VB and MUM contributed to research and to the writing of the manuscript; MC performed miQPCR, northern blot, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.Table S1 - Detailed list of degradation-regulated probes as detected by microarray. The Table contains the list of individual probes identified as regulated (i.e. for displaying fold changes of ± 2) in the microarray experiments. Data sets were correlated as indicate in the legend of Figure Click here for file