A method for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves

A method is described for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves of two higher plant species. Sodium dodecyl sulfate (1%) and 25 mM magnesium ions are required to inhibit ribonuclease action during RNA purification by phenol deproteinization. The ethanol-precipitated RNA product, including 23s chloroplast ribosomal RNA, is completely stable during electrophoresis in the absence of magnesium ions, even in the presence of EDTA. The $\text{in}$$\text{vivo}$ mole fraction of chloroplast ribosomes relative to cytoplasmic ribosomes is estimated. Bentonite is shown to cause preferential losses of chloroplast RNA during extraction.     

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

Isolating intact chloroplasts from small Arabidopsis samples for proteomic studies

We have established a method for the isolation of chloroplasts from Arabidopsis thaliana that allows proteomic studies in the context of biotic stress with small amounts of starting material. Employing a 50% Percoll layer to separate crude filtrates, the required leaf material was reduced to 2-3 g, yielding more than 300 μg of chloroplast proteins. The quality of this fraction was confirmed by immunological, enzymatic, and gel-based assays. This protocol provides intact chloroplasts from Arabidopsis plants with a high degree of integrity and purity as well as sufficient protein recovery, thereby enabling studies of plant-herbivore or plant-pathogen interactions.     

Chemical cross-linking of protein to RNA within intact ribosomal subunits from Escherichia coli.

Summary Bifunctional reagents, namely bis-(2-chloroethyl)-amine (nitrogen mustard) and activated esters of 3-(2-bromo-3-oxobutane-1-sulphonyl)-propionic acid (bromo-ketone reagent) are used to cross-link protein to RNA within intact ribosomal subunits. The cross-linked proteins are analysed on two different two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage is demonstrated by isolating cross-linked protein-oligonucleotide complexes from subunits containing ³²P-labelled RNA. Proteins S3, S4, S5, S9/S11 and S13 from the 30S subunit, and proteins L1 and L2 from the 50S subunit were cross-linked to RNA by the nitrogen mustard, together with a number of other so far unresolved proteins. Correspondingly S3, S4, S7, S9/S11, and L2 were cross-linked by the bromoketone reagent, although in lower yield. The reagents should prove useful for topographical studies on ribosomal subunits, and arguments are presented favouring the use of non-cleavable and relatively non-specific RNA-protein cross-linking reagents for such studies.     

Extraction of high-quality RNA from pancreatic tissues for gene expression studies

Extracting RNA from pancreatic tissue is notoriously challenging because of the organ's high RNase content. Standard methods using TriPure or TRIzol classically yield RNA of sufficient quality for routine gene expression analysis but not for microarray or deep sequencing analysis. Here we developed a simple method to extract high-quality RNA from mouse pancreas. Our method uses an RNase inhibitor and combines different protocols using guanidium thiocyanate–phenol extraction. It enables reproducible isolation of RNA with an RNA integrity number around 9.     

Eukaryogenesis: a model derivated from ribosomal RNA molecular phylogenise]

We have undertaken the construction of a broad molecular phylogeny of protists through the comparison of 28S rRNA molecules. The sequences from several major protistan phyla were aligned and combined with a broad database of metazoans, metaphytes, fungi and bacteria and we have derived dendrograms from both distance matrix and parsimony methods. In agreement with classical systematics, a number of monophyletic groups separated by large evolutionary distances were observed (those of the ciliates, the chlorophytes, etc.). From this analysis, several inferences on the eukaryogenesis can be made among which the ancient origin of the cytoskeleton, the late occurrence of the chloroplastic endosymbiosis and the simultaneous emergence of the triploblastic and diploblastic metazoan patterns.     

Sequence studies on 16S ribosomal RNA from a blue-green alga

Summary The 16S ribosomal RNA of the blue green algaAnacystis nidulans has been characterized in terms of the oligomers generated by digestion with T1 ribonuclease.A. nidulans by this criterion is definitely a procaryote; being no more distant from Bacilli or Enterics than the latter two are from one another.A. nidulans appears to be somewhat more closely related to the Bacilli than to the Enterics.This is contribution III in a series entitled Procaryote phylogeny.     

Wireless bioreactor for anaerobic cultivation of bacteria

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adosrbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed and the anaerobic condition is achieved with gas pack. Although the technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria, it provides a convenient way to bypass the cumbersome operation in anaerobic chamber for facultative anaerobic bacteria. Such a cultivation strategy is demonstrated with Escherichia coli with different carbon sources and hydrogen as energy source.     

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates, Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species. Correspondence to: J. M. Bautista     

Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies

We developed a simple technique for the high-yield extraction of purified DNA from mixed populations of natural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecular biological approaches to the study of microbial communities in nature. The microorganisms from near-shore marine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-mum-pore-size fluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNA was extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100 degrees C for 1.5 to 2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNA was purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresis showed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA was sufficiently pure to allow complete digestion by the restriction endonuclease Sau3AI and ligation to vector DNA. In a sample in which the extracted DNA was quantified by binding to the dye Hoechst H33258, DNA was quantitatively extracted, and 45% of the initially extracted DNA was recovered after purification. Final yields were a few micrograms of DNA per liter of seawater and were roughly 25 to 50% of the total bacterial DNA in the sample. Alternatives to the initial harvest by filtration method, including continuous-flow centrifugation and thin-channel or hollow-fiber concentration followed by centrifugation, were less efficient than filtration in terms of both time and yield, largely because of the difficulty of centrifuging the very small bacteria typical of marine plankton. These methods were judged to be less appropriate for studies of natural populations as they impose a strong selection for the larger bacteria.     

Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.     

Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples.

Formalin-fixed archival samples are known to be poor materials for molecular biological applications. We conducted a series of experiments to understand the alterations in RNA in fixed tissue. We found that formalin-fixed tissue was resistant to solubilization by chaotropic agents. However, proteinase K completely solubilized the fixed tissue and enabled the extraction of almost the same amount of RNA as from a fresh sample. The extracted RNA did not show apparent degradation. However, as reported, successful PCR amplification was limited to short targets. The nature of such 'fixed' RNA was analyzed using synthetic homo-oligo RNAs. The heterogeneous increase in molecular weight of the RNAs, measured by MALDI-TOF mass spectrometry, showed that all four bases showed addition of mono-methylol (-CH(2)OH) groups at various rates. The modification rate varied from 40% for adenine to 4% for uracil. In addition, some adenines underwent dimerization through methylene bridging. The majority of the methylol groups, however, could be removed from bases by simply elevating the temperature in formalin-free buffer. This demodification proved effective in restoring the template activity of RNA from fixed tissue. The improvement in PCR results suggested that more than half of the modification was removed by this demodification.     

Refined molecular weights for phage, viral and ribosomal RNA.

The RNAs of the Escherichia coli bacteriophages MS2 and Qbeta as well as E. coli 16S ribosomal RNA were examined under identical conditions by electron microscopy using the protein-free benzyldimethylalkylammonium chloride (BAC) spreading technique. From the contour length ratios of the RNAs and the known number of nucleotides for MS2, the chain lengths for Qbeta RNA and 16S RNA were found to be 4790 +/- 150 and 1645 +/- 55 nucleotides. Correcting for the base composition of Qbeta RNA the molecular weight of the Na salt of this RNA is (1.64 +/- 0.06) . 10(6) daltons. Since published values on the relative lengths of Qbeta RNA and several other homogeneous RNAs (E. coli 23S rRNA, E. Coli bacteriophage R17 and f2 RNAs, Pseudomonas aeruginosa phage PP7 RNA and Newcastle disease virus RNA) are available, we are able to calculate the approximate number of nucleotides for these useful standards.     

Nucleotide sequence of 5S ribosomal RNA from Lingula anatina. A study on the molecular evolution of 5S ribosomal RNA from a living fossil

By chromatography on columns of DEAE-Sephadex A-50 and Sephadex G-100, and electrophoresis on polyacrylamide gel, 5S rRNA was purified from a low-molecular-weight RNA fraction extracted from the total tissues of Lingula anatina. Complete digests of the 5S rRNA with RNase T1 [EC 3.1.4.8] and pancreatic RNase [EC 3.1.4.22] were sequenced by conventional column chromatography procedures. The nucleotide sequence of this RNA was determined mainly by a chemical method for sequencing the RNA 3' end-labeled with 32P (1), with the complement of the oligonucleotide catalog obtained by the complete RNase digestions of the RNA. By comparing the sequences of several invertebrate, vertebrate, and Chlorella 5S rRNAs, a phylogenic tree of the rRNAs was constructed and the time of divergence of Lingula was estimated.     

The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review

This paper analyses the research progress in the use of molecular techniques based on ribosomal RNA and DNA (rRNA/rDNA) for rumen microbial ecosystem since first literature by Stahl et al. (1988). Because rumen microbial populations could be under-estimated by adopting the traditional techniques such as roll-tube technique or most-probable-number estimates, modern molecular techniques based on 16S/18S rRNA/rDNA can be used to more accurately provide molecular characterization, microbe populations and classification scheme than traditional methods. Phylogenetic-group-specific probes can be used to hybridize samples for detecting and quantifying of rumen microbes. But, competitive-PCR and real-time PCR can more sensitively quantify rumen microbes than hybridization. Molecular fingerprinting techniques including both denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and restriction fragment length polymorphisms (RFLP) can used to explore diversity of bacteria, protozoa and fungi in the rumen ecosystem. By constructing clone libraries of 16S/18S rRNA/rDNA of rumen microbes, more new microbes can be discovered and identified. For fungi, internal transcribed spacers (ITS) of fungi are better than 18S rRNA/rDNA for discriminating operational taxonomic units. In conclusion, 16S/18S rRNA/rDNA procedures have been used with success in rumen microbes and are quickly gaining acceptance for studying rumen microbial ecosystem, and will become useful methods for rumen ecology research. However, molecular techniques based on 16S/18S rRNA/rDNA don't preclude classical and traditional microbiological techniques. It should used together to acquire accurate and satisfactory results.     

Comparative studies on bacterial 5S ribosomal RNA

The 5S rRNAs of Escherichia coli, Bacillus stearothermophilus, and B. subtilis were isolated and their molecular conformation examined. All three 5S rRNAs were similar with regard to nucleotide chain length, base composition and general configuration. Several major differences were apparent between the secondary and tertiary conformations of the 5S rRNA of E. coli and the genus Bacillus. Only minor differences were noted between those from the two Bacillus species. Each 5S rRNA species had a different 5′-terminal nucleotide: E. coli-U; B. stearothermophilus-C; B. subtilis-G.