Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.

Introduction to Molecular Analysis of Ectomycorrhizal Communities

A number of methods are available for those researchers considering the addition of molecular analyses of ectomycorrhizal (EcM) fungi to their research projects and weighing the various approaches they might take. Analyzing natural EcM fungal communities has traditionally been a highly skilled, time-consuming process relying heavily on exacting morphological characterization of EcM root tips. Increasingly powerful molecular methods for analyzing EcM communities make this area of research available to a much wider range of researchers. Ecologists can gain from the body of work characterizing EcM while avoiding the requirement for exceptional expertise by carefully combining elements of traditional methods with the more recent molecular approaches. A cursory morphological analysis can yield a traditional quantification of EcM fungi based on tip numbers, a unit with functional and historical significance. Ectomycorrhizal root DNA extracts may then be analyzed with molecular methods widely used for characterizing microbiota. These range from methods applicable only to the simple mixes resulting from careful morphotyping, to community-oriented methods that identify many types in mixed samples as well as provide an estimate of their relative abundances. Extramatrical hyphae in bulk soil can also be more effectively studied, extending characterization of EcM fungal communities beyond the rhizoplane. The trend toward techniques permitting larger sample sets without prohibitive labor and time requirements will also permit us to more frequently address the issues of spatial and temporal variability and better characterize the roles of EcM fungi at multiple scales.

土壤中Frankia菌RNA提取

土壤中Ｆｒａｎｋｉａ菌ＲＮＡ提取王育英，林建群，郭秀荣，李彤，张忠泽（中国科学院沈阳应用生态研究所，１１００１５）Ｆｒａｎｋｉａ非亚科共生固氮是一类重要的固氮资源。对Ｆｒａｎｋｉａ共生生物学、生态学等特性曾进行过广泛的研究。８ｔ０年代末，Ｆｒａｎｋｉ...

Self-assembly of single-stranded RNA on carbon nanotube: Polyadenylic acid to form a duplex structure

All messenger-RNA (mRNA) molecules in eukaryotic cells have a polyadenylic acid [poly (rA)] tail at the 3'-end and human poly (rA) polymerase (PAP) has been considered as a tumor-specific target. A ligand that is capable of recognizing and binding to the poly(M) tail of mRNA might interfere with the full processing of mRNA by PAP and can be a potential therapeutic agent. We report here for the first time that single-walled carbon nanotubes (SWNTs) can cause single-stranded poly (M) to self-structure and form a duplex structure, which is studied by UV melting, atomic force microscopy, circular dichroism spectroscopy, and NMR spectrometry.

Targeted RNA interference of cyclin A(2) mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells

Cyclin A(2) plays critical role in DNA replication, transcription, and cell cycle regulation. Its overexpression has been detected and related to many types of cancers including leukemia, suggesting that suppression of cyclin A(2) would be an attractive strategy to prevent tumor progression. Herein, we apply functionalized single wall carbon nanotubes (f-SWNTs) to carry small interfering RNA (siRNA) into K562 cells and determine whether inhibition of cyclin A(2) would be a potential therapeutic target for chronic myelogenous leukemia.

Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

Particle-attached and free-living bacterial communities in a contrasting marine environment: Victoria Harbor, Hong Kong

Diversity of particle-attached and free-living marine bacteria in Victoria Harbor, Hong Kong, and its adjacent coastal and estuarial environments was investigated using DNA fingerprinting and clone library analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes showed that bacterial communities in three stations of Victoria Harbor were similar, but differed from those in adjacent coastal and estuarine stations. Particle-attached and free-living bacterial community composition differed in the Victoria Harbor area. DNA sequencing of 28 bands from DGGE gel showed Alphaproteobacteria was the most abundant group, followed by the Bacteroidetes, and other Proteobacteria. Bacterial species richness (number of DGGE bands) differed among stations and populations (particle-attached and free-living; bottom and surface). BIOENV analysis indicated that the concentrations of suspended solids were the major contributing parameter for the spatial variation of total bacterial community structure. Samples from representative stations were selected for clone library (548 clones) construction and their phylogenetic distributions were similar to those of sequences from DGGE. Approximately 80% of clones were affiliated to Proteobacteria, Bacteroidetes and Cyanobacteria. The possible influences of dynamic pollution and hydrological conditions in the Victoria Harbor area on the particle-attached and free-living bacterial community structures were discussed.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Using the ribosomal internal transcribed spacer (ITS) as a complement marker for species identification of red macroalgae

Barcodes based on mitochondrial cytochrome oxidase (mtDNA CO1) sequences are being used for broad taxonomic groups of animals with demonstrated success in species identification and cryptic species discovery, but it has become clear that complementation by a nuclear marker system is necessary, in particular for the barcoding of plants. Here, we propose the nuclear internal transcribed spacer (ITS) as a potentially usable and complementary marker for species identification of red macroalgae, as well as present a primary workflow for species barcoding. Data show that for most red macroalgal genera (except members of the family Delesseriaceae), the size of ITS region ranges from 600 to 1200 bp, and contains enough variation to generate unique identifiers at either the species or genus levels. Consistent with previous studies, we found that the ITS sequence can resolve closely related species with the same fidelity as mtDNA CO1. Significantly, we confirmed that length polymorphism in the ITS region (including 5.8S rRNA gene) can be utilized as a character to discriminate red macroalgal species. As a complementary marker, the verifiable nuclear ITS region can speed routine identification and the detection of species, advance ecological and taxonomic inquiry, and permit rapid and accurate analysis of red macroalgae.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.

Complete mitochondrial genome of the sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea): The first representative from the subclass Aspidochirotacea with the echinoderm ground pattern

Complete mitochondrial genome plays an important role in the accurate revelation of phylogenetic relationships among metazoans. Here we present the complete mitochondrial genome sequence from a sea cucumber Apostichopus japonicus (Echinodermata: Holothuroidea), which is the first representative from the subclass Aspidochirotacea. The mitochondrial genome of A. japonicus is 16,096 bp in length. The heavy strand consists of 31.8% A, 20.2% C, 17.9% G, and 30.1% T bases (AT skew = 0.027: GC skew = 0.062). It contains thirteen protein-coding genes (PCGs), twenty-two transfer RNA genes, and two ribosomal RNA genes. There are a total of 3793 codons in all thirteen mitochondrial PCGs, excluding incomplete termination codons. The most frequently used amino acid is Leu (15.77%), followed by Set (9.73%), Met (8.62%), Phe (7.94%), and Ala (7.28%). Intergenetic regions in the mitochondrial genome of A. japonicus are 839 bp in total, with three relatively large regions of Unassigned Sequences (UAS) greater than 100 bp. The gene order of A. japonicus is identical to that observed in the five studied sea urchins, which confirms that the gene order shared by the two classes (Holothuroidea and Echinoidea) is a ground pattern of echinoderm mitochondrial genomes. Bayesian tree based on the cob gene supports the following relationship: (outgroup, (Crinoids, (Asteroids, Ophiuroids, (Echinoids, Holothuroids)))). (C) 2009 Elsevier B.V. All rights reserved.

Identification of peptides that bind with thymidylate synthase RNA using mRNA display technique

Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library (>10(13) different sequences). The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

Molecular phylogeny and species identification of pufferfish of the genus Takifugu (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships and species identification of pufferfishes of the genus Takifugu were examined by use of randomly amplified polymorphic DNA (RAPD) and sequencing of the amplified partial mitochondrial 16S ribosomal RNA genes. Amplifications with 200 ten-base primers under predetermined optimal reaction conditions yielded 1962 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances between 5 species of Takifugu and Lagocephalus spadiceus as the outgroup were calculated from the presence or absence of the amplified fragments. Approximately 572 bp of the 16S ribosonial RNA gene was amplified, using universal primers, and used to determine the genetic distance values. Topological phylogenic trees for the 5 species of Takifugu and outgroup were generated from neighbor-joining analysis based on the data set of RAPD analysis and sequences of mitochondrial 16S rDNA. The genetic distance between Takifugu rubripes and Takifugu pseudommus was almost the same as that between individuals within cacti species, but much smaller than that between T. rubripes, T. pseudommus, and the other species. The molecular data gathered from both analysis of mitochondria and nuclear DNA strongly indicated that T. rubripes and T. pseudommus should be regarded as the same species. A fragment of approximately 900 bp was amplified from the genome of all 26 T. pseudommus individuals examined and 4 individuals of intermediate varieties between T. rubripes and T. pseudommus. Of the 32 T. rubripes individuals, only 3 had the amplified fragment. These results suggest that this fragment may be useful in distinguishing between T. rubripes and T. pseudommus.

The complete mitochondrial genomes of two common shrimps (Litopenaeus vannamei and Fenneropenaeus chinensis) and their phylogenomic considerations

Complete mitochondrial genomes have proven extremely valuable in helping to understand the evolutionary relationships among metazoans. However, uneven taxon sampling may lead to unclear or even erroneous phylogenetic topologies. The decapod crustaceans are relatively well-sampled, but sampling is still uneven within this group. We have sequenced the mitochondrial genomes of two shrimps Litopenaeus vannamei and Fenneropenaeus chinensis. As seen in other metazoans, the genomes contain a standard set of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and an AT-rich non-coding region. The gene arrangements are consistent with the pancrustacean ground pattern. Both the pattern of gene rearrangements and phylogenomic analyses using concatenated nucleic acid and amino acid sequences of the 13 mitochondrial protein-coding genes strengthened the support that Caridea and Palinura are primitive members of Pleocyemata. These sequences, in combination with two previously published penaeid mitochondrial genomes, suggest that genera within the family Penaeidae have the following relationship: (((Penaeits + Fenneropenaett.) + Litopeiiaelts) + Marsupenaeus). The analyses of nucleic acid and amino acid sequences of the mitochondrial genomes also strongly support the monophyly of Penaeidae, Brachyura and Pleocyemata. In addition, the analyses of the average Ka/Ks in the 13 mitochondrial protein-coding genes of penaeid shrimps indicated a strong purifying selection within this group.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. (c) 2007 Elsevier Ltd. All rights reserved.