996 resultados para RNA isolation


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This paper reviews a study to examine the effects on lip reading performance of word position within a sentence.

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Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

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Lactoperoxidase (LP) was isolated from whey protein by cation-exchange using Carboxymethyl resin (CM-25C) and Sulphopropyl Toyopearl resin (SP-650C). Both batch and column procedures were employed and the adsorption capacities and extraction efficiencies were compared. The resin bed volume to whey volume ratios were 0.96:1.0 for CM-25C and ≤ 0.64:1.0 for SP-650 indicating higher adsorption capacity of SP-650 compared to CM-25C. The effluent LP activity depended on both the enzyme activity in the whey and the amount of whey loaded on the column within the saturation limits of the resin. The percentage recovery was high below the saturation point and fell off rapidly with over-saturation. While effective recovery was achieved with column extraction procedures, the recovery was poor in batch procedures. The whey-resin contact time had little impact on the enzyme adsorption. SDS PAGE and HPLC analyses were also carried out, the purity was examined and the proteins characterised in terms of molecular weights. Reversed phase HPLC provided clear distinction of the LP and lactoferrin (LF) peaks. The enzyme purity was higher in column effluents compared to batch effluents, judged on the basis of the clarity of the gel bands and the resolved peaks in HPLC chromatograms.

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Several tissue types of Lupinus albus L. were investigated as sources for the isolation of protoplasts. Cotyledons from in vitro seedlings were found to yield the highest number of protoplasts compared with leaves, hypocotyls and roots. A combination of the protoplast isolation enzymes, cellulase and Pectolyase Y23, was capable of releasing the highest number of protoplasts compared with a combination of cellulase and Macerase. Protoplast yield increased with increasing cotyledon age but was accompanied by a progressive decline in protoplast viability. The optimal combination of protoplast yield and viability occurred when the protoplasts were isolated from 14- to 18-day-old cotyledons. The ratio between the volume of enzyme solution and the tissue biomass did not affect the protoplast production significantly. This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 x 10(6) protoplasts per gram of fresh tissue was obtainable.

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We have obtained a single spore isolate of Pasteuria penetrans, derived by allowing a single spore to attach to a second-stage juvenile (J2) of the root-knot nematode Meloidogyne javanica. By analysing DNA sequences at three different loci we have obtained evidence that the isolate is, indeed, genetically pure. We compared the ability of the single spore isolate and the parent population from which it was selected to attach to and parasitise both the original population of M. javanica on which it was isolated and a single egg mass line derived from it. There was no difference in the attachment of spores of the single spore isolate to juveniles compared to the parental population, although there were higher numbers of both attaching to J2 of the single egg mass line compared to its parental population. Judging from the numbers of egg masses and Pasteuria-infected females, the single spore isolate was less pathogenic to the parental population of M. javanica than was the parental spore population.

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Here we describe a novel, inexpensive and simple method for preserving RNA that reduces handling stress in aquatic invertebrates following ecotoxicogenomic experimentation. The application of the method is based on transcriptomic experiments conducted on Daphnia magna, but may easily be applied on a range of other aquatic organisms of a particular size with e.g. amphipod Gammarus pulex representing an upper size limit. We explain in detail how to apply this new method, named the "Cylindrical Sieve (CS) system", and highlight its advantages and disadvantages.

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IMAC can be used to selectively enrich phosphopeptides from complex peptide mixtures, but co-retention of acidic peptides together with the failure to retain some phosphopeptides restricts the general utility of the method. In this study Fe(III)-IMAC was qualitatively and quantitatively assessed using a panel of phosphopeptides, both synthetic and derived from proteolysis of known phosphoproteins, to identify the causes of success and failure in the application of this technique. Here we demonstrate that, as expected, peptides with a more acidic amino acid content are generally more efficiently purified and detected by MALDI-MS after Fe(III)-IMAC than those with a more basic content. Modulating the loading buffer used for Fe(III)-IMAC significantly affects phosphopeptide binding and suggests that conformational factors that lead to steric hindrance and reduced accessibility to the phosphate are important. The use of 1,1,1,3,3,3-hexafluoroisopropanol is shown here to significantly improve Fe(III)-IMAC enrichment and subsequent detection of phosphopeptides by MALDI-MS.

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Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphospborylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5' end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

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The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 313 (VTg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV ere has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 313 peptides has now been determined, and the role of the FMDV ere (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

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The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5±3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.

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Archaea, the third domain of life, were long thought to be limited to environmental extremes. However, the discovery of archaeal 16S rRNA gene sequences in water, sediment and soil samples has called into question the notion of Archaea as obligate extremophiles. Until now none of these novel Archaea has been brought into culture, a critical step for discovering their ecological roles. We have cultivated three novel halophilic Archaea (haloarchaea) genotypes from sediments in which the pore-water salinity was close to that of seawater. All previously reported haloarchaeal isolates are obligate extreme halophiles requiring at least 9% w/v NaCl for growth and are typically the dominant heterotrophic organisms in salt and soda lakes, salt deposits and salterns. Two of these three newly isolated genotypes have lower requirements for salt than previously cultured haloarchaea and are capable of slow growth at seawater salinity (2.5% w/v NaCl). Our data reveal the existence of Archaea that can grow in non-extreme conditions and of a diverse community of haloarchaea existing in coastal salt marsh sediments. Our findings suggest that the ecological range of these physiologically versatile prokaryotes is much wider than previously supposed.

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This study reports on the influence of critical isolation factors on the subsequent culture of protoplasts of Lupinus albus L. Protoplasts were isolated from in vitro seedling cotyledons of five early maturing accessions in which protoplast yields and division frequencies appeared to be correlated as a high protoplast yield corresponded with a high division frequency. The overall difference among the accessions for mitosis was non- significant, although the highest yield and division frequency were observed in accession LA132, with Alban giving a significantly lower level. Accession Lucrop produced the lowest number of protoplasts, all of which collapsed during culture. Of the enzyme types used for tissue maceration, Pectolyase Y23, was significantly inferior to Macerase in terms of giving way to mitosis. The extent of division in Macerase- isolated protoplast population was 266% higher than that in the Pectolyase Y23- isolated one. The physiological maturity level of the explant, expressed in terms of developmental age, was optimal when 14 - 18- day- old seedling cotyledons were used for protoplast production and culture, rather than more mature ones, despite higher protoplast yields in the latter. On K8p medium, the protoplast division frequency was 129% greater when 18- day- old seedling cotyledons were used, than that with any other treatment. This work on protoplast culture of the potentially important lupin species, which is a pulse rich in dietary protein, oil and fibre, allows a further understanding of the biology, with an aim to advance lupin biotechnology.