Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.     

Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit

A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol in an extraction buffer in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable for a variety of plant tissues.     

Isolation of RNA from apple skin

A method has been developed for the isolation of RNA from apple skin. The method involves an adaptation of the Manning (1991) method and includes a high-salt extraction step and a final purification step through a CsCl cushion. The RNA isolated was of high quality and produced good hybridization signals in northern blot analyses.     

Isolation of RNA polymerases from the water mold Achlya

The DNA-dependent RNA polymerases I, II, and III (ribonucleosidetriphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) from Achlya ambisexualis E87 (male), have been isolated. The highly purified RNA polymerase I was found to be composed of polypeptides with the following molecular weights (·10^-4): 18.5, 14, 11.8, 7.3, 6.1, 4.9, 4.4, 2.8. RNA polymerase II showed a 400-fold higher resistance against -amanitin than mammalian or higher plant RNA polymerase II.     

The isolation of RNA from raspberry (Rubus idaeus) fruit

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.     

Isolation of RNA from floral tissue ofRumex acetosa (Sorrel)

Flower tissue ofRumex acetosa was previously intractable for the isolation of RNA using standard methods, due probably to its high level of polysaccharides. Extraction at low pH, precipitation of polysaccharides with potassium acetate followed by precipitation of RNA with lithium chloride yielded high-quality RNA that was suitable for northern hybridisation,in-vitro translation, poly(A)⁺ RNA selection, and subsequent cDNA synthesis.     

DNA-dependent RNA polymerases from Drosophila melanogaster adults: Isolation and partial characterization

In preparation for the isolation and biochemical characterization of putative RNA polymerase mutants, DNA-dependent RNA polymerases of Drosophila melanogaster adults were isolated and partially characterized. Approximately 70% of the female adult RNA polymerase is located in ovaries. Multiple forms of ovarian RNA polymerases I and II are separable by DEAE-Sephadex chromatography. The two forms of RNA polymerase II differ in ammonium sulfate optima. RNA polymerase IIA is more active with double-stranded DNA as template, whereas RNA polymerase IIB transcribes single-stranded DNA most efficiently. Rechromatography of RNA polymerase IIA on DEAE-Sephadex results in the loss of ability of this form to transcribed double-stranded DNA most efficiently. Ovariectomized carcasses have two forms of RNA polymerase I and one form of RNA polymerase II and each transcribes single-stranded DNA most efficiently. As judged by gel filtration chromatography, female adult extracts have forms of RNA polymerase II that differ in molecular weight and template preference.Supported by Grants GM23456 from the NIH and 11259 from the City University Research Foundation.     

A simple procedure for the isolation of high quality RNA from ripening banana fruit

Isolation of high quality RNA from ripening banana fruit tissue is difficult due to high levels of polysaccharides and other substances that interfere when using conventional procedures for RNA isolation. These substances not only decrease the yield but the quality of RNA is almost unusable. We describe here a simple RNA procedure that effectively removes these contaminating substances without affecting the yield. Following this procedure, we routinely obtained 80–150 μg of total RNA per g fresh tissue. The RNA is of good quality and suitable for northern analysis, RT-PCR and cDNA library construction. NBRI publication No. 488(NS).     

迟钝爱德华氏菌RNA提取及痕量基因组DNA去除方法探究

迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。     

Production of single-cell oil from prickly-pear juice fermentation by Cryptococcus curvatus grown in batch culture

The biomass of Cryptococcus curvatus, an oleaginous yeast, reached 11 g/l and accumulated 46% (w/w) lipid when grown for 35 h in batch culture on diluted (25%) prickly-pear juice. The C:N ratio of the juice was about 50 g/g. The efficiency of substrate conversion was 0.48 g/g for biomass and 0.22 g/g for lipids. The extracted lipids were mainly oleic (18:1) and palmitic (16:0) acids and the quality of pipid approached that of palm oil.M. Hassan was and P.J. Blanc, A. Pareilleux and G. Goma are with the Département de Génie Biochimique et Alimentaire, URA-CNRS 544, Institut National des Sciences Appliquées. Complexe Scientifique de Rangueil, F-31077 Toulouse Cedex France; M. Hassan is now with the Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh.     

Isolating high-quality RNA from mangroves without liquid nitrogen

Mangroves form unique communities in tropical coastal regions and tidal lowlands. Isolating RNA from mangrove leaves is difficult because of high amounts of secondary metabolites and polysaccharides. Conventional extraction methods produce poor-quality mangrove RNA. We present a simple, fast, and convenient protocol for isolating RNA from 5 mangrove species:Aegiceras corniculatum, Bruguiera gymnorrhiza, Ceriops tagal, Kandelia candel, andSonneratia apetala. Isolating RNA from other mangrove species is also possible. Obtained RNA was of high quality and used in an RT-PCR reaction that amplified 0.6 kb of theA. corniculatum CPI-1 gene.     

A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites

A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure^™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A_260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen^™ reagent and of PCR products with the PicoGreen^™ reagent.     

Characterization of ethylene production in developing strawberry fruit

Ethylene production, ACC content, and ACC oxidase activity were determined in strawberry fruit harvested at different stages of development and in fruit harvested green and developed in vitro in solutions containing sucrose. In fruit harvested at progressive stages of development from green through full ripe, ethylene production and ACC oxidase activity decreased whereas ACC content increased between the white and pink stages. Fruit detached at the green stage and developed to full ripe by immersion of the cut pedicel in sucrose solutions exhibited an increase in ACC content, decreased ethylene production, and no change in ACC oxidase activity. Detached green fruit provided with sucrose containing 0.5 mM silver (STS) had elevated ethylene production and more ACC oxidase activity than did fruit incubated without the silver salt. Green fruit provided with sucrose containing 1 mM ACC showed markedly increased ACC content, ACC oxidase activity, and ethylene production. These increases were noted following 4 days incubation in ACC, and were more pronounced after 11 days, at which time fruit of all treatments had attained a full-ripe stage of development. Calyx tissue exhibited more ACC oxidase activity, less ACC content, and similar ethylene production compared with receptacle tissue. ACC synthase could not be detected in fruit harvested at different developmental stages or in fruit detached and developed in vitro.abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - HQS 8-hydroxyquinoline hemisulfate - SAM S-adenosyl methionine - STS silver thiosulfate     

Host fruit chemical stimuli eliciting distinct ovipositional responses from sibling species of Rhagoletis fruit flies

Laboratory experiments tested whether two economically-important sibling species of tephritid fruit flies have evolved distinct egg-laying responses to chemical stimuli on the fruits of their respective hostplants. The egg-laying preferences displayed by apple maggot flies, R. pomonella, and blueberry maggot flies, R. mendax, on artificial fruits treated with apple and blueberry extract paralleled their egg-laying responses to whole apples and blueberries. R. pomonella flies laid more eggs than R. mendax flies in artificial fruits treated with extract from ripe McIntosh apples, and vice versa for artificial fruits treated with extract from ripe Bluehaven blueberries. Furthermore, both species laid more eggs in artificial fruits treated with extract from their respective host fruits than control artificial fruits which were not treated with fruit extract. Prior electroantennogram recordings from R. mendax and R. pomonella flies exposed to volatiles from pentane extracts of apples and blueberries indicate that the antennal sensitivity of both species is selectively tuned to their respective host fruit odors. This differentiation in their olfactory responses to fruit odors could be important in mediating their distinct ovipositional responses to blueberry and apple fruits. Extract from unripe McIntosh apples also elicited egg laying by R. pomonella flies, however, artificial fruits treated with unripe apple extract received 1.9 times fewer eggs than those treated with ripe apple extract. Moreover, the numbers of R. pomonella ovipositor punctures and eggs placed in wax artificial fruits were increased when the artificial fruits were treated with a blend of 7 identified apple esters. Black coloration on these artificial fruits and the presence of apple esters had a synergistic effect on the egg-laying behavior of R. pomonella flies, which caused them to lay substantially more eggs per black fruit than white fruit treated with the same concentration of apple esters. In summary, our results indicate that the egg-laying responses of R. pomonella flies are mediated by the integration of information from fruit chemical and visual cues, and that R. mendax and R. pomonella flies have evolved divergent egg-laying responses to chemical stimuli on the fruits of their respective hostplants. These findings are discussed in the context of other studies on plant compounds which influence the ovipositional behavior of phytophagous Diptera.

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Stimuli chimiques des pommes et des myrtilles induisant la ponte des espèces jumelles, Rhagoletis pomonella et R. mendax

Résumé Des fruits artificiels en cire traités avec des extraits de fruits ont provoqué chez les espèces jumelles de R. mendax (Curran) et R. pomonella (Walsh) des réactions de ponte différentes suivant les stimulations chimiques par les fruits. Le comportement de ponte sur des fruits artificiels traités avec des extraits au pentane des myrtilles mûres (Vaccinium corymbosum L.) et de pommes mûres (Malus pumila Miller = Pyrus malus L.), est le même que sur des fruits naturels, ce qui montre que la réponse aux stimulations chimiques provenant du fruit constitue un aspect important de la reconnaissance de l'hôte. R. pomonella pond plus d'ufs que R. mendax sur les fruits artificiels traités à l'extrait de pommes mûres; c'est l'inverse pour les fruits traités aux extraits de myrtille. Les fruits artificiels traités avec des pommes ou des myrtilles provoquent la ponte de R. pomonella, tandis que les myrtilles mûres seules provoquent la ponte de R. mendax. Les extraits de pommes vertes stimulent la ponte de R. pomonella mais elle est alors 2 fois plus faible qu'avec des extraits de pommes mûres. Un mélange de 7 esters identifiés dans l'extrait de pomme induit aussi la ponte de R. pomonella. Le nombre de piqûres de tarièresfli dans les fruits artificiels en cire et le nombre d'ufs par fruit ont été augmentés par addition d'esters de pommes à des fruits blancs ou noirs. La couleur des fruits artificiels influence aussi la réaction de ponte de R. pomonella; la fréquence des piqûres de tarière contenant un uf et le nombre d'ufs par fruit étaient significativement plus élevés sur les fruits noirs que sur les fruits blancs traités avec la même concentration d'esters de pomme. Les fruits artificiels noirs traités avec la concentration la plus stimulante d'esters de pommes ont reçu 2, 3 fois plus d'ufs que les fruits blancs avec les mêmes concentrations en esters. Ces résultats montrent que les esters de pomme et la couleur noire stimulent synergiquement la ponte de R. pomonella sur des fruits artificiels.

     

A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 °C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.Communicated by G. Antranikian     

DNA-dependent RNA polymerase II from nuclei of suspension-cultured tobacco cells

From isolated nuclei of suspension cultured cells of Nicotiana tabacum. DNA-dependent RNA polymerase II (E.C. 2.7.76) has been purified to homogeneity as evidenced by polyacrylamidegel electrophoresis under non-denaturing conditions. The purified enzyme had a specific activity of more than 15 nmol min^-1·mg^-1 with denatured calf thymus DNA as template. Sodium-dodecyl-sulfate gel electrophoresis and protein highperformance liquid chromatography revealed a subunit composition of four proteins with molecular weights of 165 000, 135 000, 35 000 and 25 000 and with a stoichiometry of 1:1:2:2. The RNA polymerase did not exhibit any detectable proteinkinase activity. The 25 000 subunit binds ADP in a molar ratio of 1:1; it could not be decided whether this subunit has an ATPase activity or is merely an acceptor of ADP.Abbreviations HPLC high-performance liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate This contribution is dedicated to Professor Fritz Cramer on the occasion of his 60th birthday     

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.     

猕猴桃RNA提取与RT-PCR

为了从富含多糖和多酚等物质的猕猴桃幼叶中提取和分离出高质量的RNA,用多个不同品种的猕猴桃叶为材料,比较了3种不同的RNA提取方法所提取的总RNA。结果表明,用胍-酚酸-DEPC法提取的RNA质量最好,提取率达到682.9~780.8μg?g(FW),其R值(A260?A280)接近1.90。用所提取的RNA样品进行RT-PCR,其扩增产物在琼脂糖凝胶上出现明显清晰的扩增cDNA带,说明RNA样品在纯度和浓度上都可以满足PCR等分子生物学实验的基本要求。