Sex ratio variation and spatial segregation of the sexes in populations ofRumex acetosa andR. acetosella (Polygonaceae)

Populations ofRumex acetosa andR. acetosella were studied during two growth seasons. The ramet sex ratios ofR. acetosa were always female-biased. InR. acetosella the sex ratios expressed more variation but were mostly female-biased. In both species the sex ratios commonly varied between subpopulations reflecting a partial spatial segregation of the sexes. No marked differences between sexes in vegetative vigour were detected in either species. Interactions between sex ratios, various soil characteristics and population densities were determined. Possible mechanisms for causing biased sex ratios and partial spatial segregation of the sexes are discussed.     

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.     

In vitro organogenesis in two dioecious species,Rumex acetosella L. and R. acetosa L. (Polygonaceae)

Callus cultures were established from dioecious plant species Rumex acetosella and R. acetosa, using cotyledons, hypocotyls and stem tips of aseptically germinated seedlings as primary explants. Cultures were also established from male and female R. acetosella adult plants, starting from vegetative lateral buds. Cell division was induced using a high 2,4-D concentration, while bud induction and multiplication were stimulated on a medium with high BAP/IAA ratio. Cotyledon fragments of both species produced only rhizogenic calli. Hypocotyl-derived calli of R. acetosella produced buds, while those of R. acetosa showed no bud forming response under these conditions. Bud multiplication occurred in stem tip cultures of both species and in lateral bud cultures of R. acetosella. Calli derived from male plants produced more buds than those from female. Shoots were easily rooted using IBA, and plantlets were effectively transferred to soil. Flowering was not induced in culture. The sex of regenerated male and female plants was not altered by the culture conditions.     

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.     

Sex chromatin and nucleolar analyses inRumex acetosa L.

Summary Rumex acetosa (sorrel) is a dioecious plant with a XX/XY₁Y₂ sex chromosome system. Both the Y chromosomes are nearly entirely heterochromatic and it has been hypothesised that they can persist as chromocenters in male interphase nuclei. Using specific antibodies against 5-methylcytosine and histone H4 acetylated at terminal lysine 5, global levels of DNA methylation and histone acetylation were studied on the sex chromosomes and autosomes of both sexes. The heterochromatic Y chromosomes did not display a higher methylation level compared to the autosomes. The only prominent hypermethylation signals were found at two nucleolar organising regions located on the autosome pair V, as confirmed by in situ hybridisation with 25S rDNA probe and staining. Immunoanalysis of DNA methylation on female and male interphase nuclei neither revealed any sex-specific differences. Two active (silverpositive) nucleoli and two likely inactive nucleolar organising regions (displaying prominent methylation signals) were found in both sexes. In a fraction of nuclei isolated from leaf cells, two peripheral bodies strongly positive for 4,6-diamidino-2-phenylindole were observed only in males, never in females. These heterochromatin regions were depleted in histone H4 acetylation at terminal lysine 5 and corresponded, according to in situ hybridisation with a Y-chromosome-specific repetitive probe, to the two Y chromosomes. We conclude that the peripheral condensed bodies observed exclusively in male nuclei represent the constitutive heterochromatin of the Y chromosomes which is characterised by a substantial histone H4 underacetylation.     

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 high quality RNA from seeds and tubers of the Mexican yam bean (Pachyrhizus erosus)

Pachyrhizus erosus is a tuberous legume native to Central America that has great potential for development as a food crop. It produces both protein rich grain and starch filled tubers. There are two major limitations to its dietary use, the high levels of rotenone found in the grains and the low starch content of the tubers, both of which must be addressed, for development of the crop. The low variability of the existing gene pool of the genus limits the use of conventional plant breeding to address these problems. Genetic engineering technology is, therefore, being adopted. For this purpose, an efficient means of RNA isolation from yam bean tissues was developed. The quality of RNA obtained by this method was tested byin vitro translation and was sufficient for use in RT-PCR.     

Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use

A new procedure is described which enables gram quantities of adult Onchocerca tissue to be isolated from frozen connective tissue nodules, thus minimizing the risk of enzymatic degradation. Bovine connective tissue nodules containing adult Onchocerca gibsoni worms were obtained from Australia frozen at −70°C and sectioned while still frozen into 3 mm thick slabs. The sections were thawed immediately before use, worm segments removed, rinsed, pelleted, and flash frozen in liquid nitrogen. Quality of the isolated material was demonstrated by the presence of an intact adult epicuticle as determined by electron microscopy, and by the presence of viable uterine larvae and cells. This procedure is applicable to other nodule-forming worms such as Onchocerca volvulus and is suitable for investigations which require the isolation of labile molecules or those present in minute quantities.     

Small RNA species in maize tissue

The low molecular weight RNA components of maize have been analyzed after labeling callus and leaf tissue with [³H]uridine in vitro. Electrophoresis of the isolated RNA on acrylamide slab gels reveals, apart from 5S and transfer RNA, three major and about five minor RNA species with chain lengths between 140 and 280 nucleotides. These RNA molecules are labeled as rapidly as 5S, transfer RNA, and do not represent degradation products of large ribosomal RNA molecules. Furthermore, like 5S and transfer RNA, these small RNA species are stable and show no detectable turnover within forty-eight hours. Fractionation of the tissue into crude subcellular fractions indicates a preferential association of some of the small stable RNA species with the nucleus, while others appear to be located in the cytoplasm. The low molecular weight RNA spectrum from the leaf is similar to that observed in callus, with the major small RNA species equally present in both tissues.Abbreviations tRNA transfer RNA - hnRNA heterogenous nuclear RNA - mRNA messenger RNA - scRNA small cytoplasmic RNA - snRNA small nuclear RNA     

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.     

A Method for Isolation of Total RNA from Fruit Tissues of Banana

We describe a rapid and efficient method for isolation of total RNA from banana fruit tissues. The RNA was extracted with a high ionic strength buffer at room temperature. The proteins, genomic DNA and secondary metabolites in the extract were then removed by precipitation with pre-cooled potassium acetate and repeated phenol/chloroform/isoamyl alcohol extractions. The RNA was recovered by ethanol precipitation. It was relatively free of ribonucleases and was suitable for RT-PCR and northern blot analysis. The procedure can be completed in less than 4 hours.     

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.     

Isolation of functional RNA from cactus fruit

Isolating RNA from cactus fruit is notoriously difficult because the fruit contains high amounts of secondary metabolites and polysaccharides. These form insoluble complexes with nucleic acids during extraction and can inhibit enzyme action. Our procedure allows for the extraction of RNA from finely ground tissue. The RNA we isolated was of high quality and undegraded, as gauged by spectrophotometry and electrophoresis in agarose gels. Quality was further assessed through use of the RNA in RT-PCR and northern blot analysis, indicating that it could be used to construct cDNA libraries. Using this modified protocol, 90μg of RNA was routinely obtained from 1 g of dried cactus fruit. Isolating RNA from other polysaccharide-rich fruits was also possible.     

日本结缕草总RNA提取和mRNA分离方法的优化

介绍了分别以日本结缕草根部、叶部、匍匐茎等为材料的总RNA提取的改良步骤,质量和浓度检测及其注意事项,并采用Promega公司的Poly ATract Systems Ⅲ(Z5300)试剂盒进行mRNA分离,尽管这一方法成熟可行,但仍存在洗脱体积较大,常需先沉淀浓缩后才能进行后续试验(如cDNA合成),在制备少量mRNA时回收率较低等问题。据此,对mRNA分离方法作了改良,介绍了mRNA分离的优化步骤,通过增加体系形成了一种更适合于日本结缕草mRNA分离的方法,从而为日本结缕草的分子生物学研究提供基础资料。结果证明所提取的总RNA和mRNA完整,质量较高,并应用到日本结缕草cD-NA文库的构建。     

Isolation of protoplasts from tissue fragments of Philippine cultivars of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Solieriaceae,Rhodophyta)

Protoplasts were isolated from tissue fragments (<1 mm²) of three Philippine cultivars of Kappaphycus alvarezii: the giant cultivar, cultivar L and Bohol wild type, by enzymatic dissolution of cell walls. Yields of viable protoplasts from young and old thalli (apical, middle, basal segments) were compared at various temperatures, duration of treatment and pH using eight combinations of commercial enzymes (abalone acetone powder and cellulase), and prepared extracts from fresh viscera of abalone (Haliotis asinina) and a terrestrial garden snail. Isolated protoplasts were grown in various culture media, temperatures, photoperiods and irradiance values to determine the conditions that favor germination and growth.Protoplast yields in tissues treated with commercial enzymes and the garden snail extract were lower than those obtained in tissues treated with fresh abalone extracts. Generally, the number of viable protoplasts increased with duration of enzyme treatment at 25 °C with a maximum yield of 8.2 × 10³ g⁻¹ tissue at 48 h. Yields were consistently higher in all cultivars at pH 6.1. The yields were also high from the middle segments of the giant cultivar (3.7 × 10³ g⁻¹ tissue) and Bohol wild type (4.5 × 10³ g⁻¹ tissue) treated with fresh abalone extract, and from basal segments of cultivar L and tissues treated with garden snail extract. The germination rate of protoplasts was highest (39.8%) at 25 °C and 20 μmol photon m⁻² s⁻¹, using a 12:12 light dark photoperiod. The filament was 3.7 mm long by Day 5. These findings are relevant to developing cultures from protoplasts for genetic or strain improvement of K. alvarezii cultivars.     

Extraction of High Quality of RNA and Construction of a Suppression Subtractive Hybridization (SSH) Library from Chestnut Rose (Rosa roxburghii Tratt)

Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and β-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 μg RNA g⁻¹ fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes. Qiang Xu and Xiaopeng Wen - Contribute to this work equally Revisions requested 3 November 2005; Revisions received 18 January 2006     

石斛总RNA提取方法的研究

采用TRIZOL法、异硫氰酸胍法、Tris-硼酸法和改良的RNA提取方法提取石斛的总RNA,并通过凝胶电泳、紫外分光光度法检测提取的RNA样品的品质。研究结果表明:改良的RNA提取方法提取的RNA具有28S rRNA和18S rRNA两条清晰的条带,且无降解。OD260nm/OD280nm接近2.0,具有较高的纯度。其它三种方法获得的RNA品质较差,有降解和弥散现象。将改良的RNA提取方法提取的RNA逆转录成cDNA,经RAPD扩增,出现清晰的条带,进一步证明改良的RNA提取方法提取的RNA具有很高的纯度,可以满足进一步分子生物学研究的要求。     

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.