Nucleotide Sequence Of RNA Research Articles

The Fast-Growing Strain of Hepatitis A Virus MB-7/293 (HepA-293) Adapted to HEK293 Cell Culture: Properties of Virus Production and Analysis of Genomic RNA

Hepatitis A virus (HAV) continues to cause outbreaks of viral hepatitis, which remains a socially significant disease despite existing vaccines. During the production of vaccines, HAV strains that require prolonged incubation of infected cell cultures, up to 4 weeks, are used. Several HAV strains, which are fast-growing on cell cultures, up to 7–10 days, are known. This paper presents the results of virological characterization and analysis of the genome of the HAV strain MB-7/293 (HepA-293) derived from MB-7/4647, one of the fast-growing strains, which was obtained by adapting it to HEK293 cell culture (human embryonic kidney cells). It was shown that the MB-7/293 strain significantly exceeds the parent MB-7/4647 strain in productivity when cultured on HEK293 cell culture, i.e., in the yield of the infectious virus and the virus-specific antigen. Analysis of the nucleotide sequence of full-length genomic RNA of the MB-7/293 strain showed that it differs from the genome sequence of MB-7/4647 in three nucleotide substitutions in the 5'-untranslated region and 20 nucleotide substitutions in the coding region, which lead to substitutions of ten amino acid residues in the proteins VP3, VP1, 2B and 2C. Comparative analysis of the genomic sequences of the strains MB-7/293, MB-7/4647, and other HAV strains, including the previously described fast-growing strains, allowed to reveal mutations that affect the reproductive properties of the virus.

THz vibrational spectroscopy for RNA basepair cocrystals and oligonucleotide sequences.

Fourier infrared spectrometer and X-ray diffractometer were used to detect the spectra of lab-made U:A (uracil and adenine hydrate dried at room temperature), lab-made C:G (cytosine and guanine hydrate dried at room temperature), U + A (mixture of uracil and adenine), and C + G (mixture of cytosine and guanine). The results of our testing showed that U:A did not form a eutectic, but C:G did. In order to further characterize the vibrational modes of RNA base pair crystals, the absorption spectra of 1‑methyl‑5‑bromouracil‑9‑methyladenine (abbreviated as 1M5BU:9MA) and 1‑methylcytosine‑9‑ethylguanine (abbreviated as 1MC:9EG) were calculated based on the PBC-GEBF (generalized energy-based fragmentation approach under periodic boundary conditions) method. To further study the effect of substituents on the vibrational mode of the crystal structure, the substituents of 1M5BU:9MA and 1MC:9EG were artificially removed. The results of calculation brought out that methyl and ethyl as substituents have little effect on the vibrational spectrum, but halogen atoms such as Br atom in 1M5BU:9MA has a certain influence on the spectrum. Furthermore, THz (terahertz) spectra of the RNA nucleotide sequence 5'-AUCG-3' was analyzed. In the perspective of the spectra with DNA signatures, their spectra show a great deal of similarity regardless of RNA or DNA, or the base sequence difference. This study will provide a very important information for revealing the role of RNA in the formation of biological macromolecules and its mechanism.

Alstroemeria yellow spot virus (AYSV): a new orthotospovirus species within a growing Eurasian clade

An orthotospovirus distinct from all other orthotospoviruses was isolated from naturally infected alstroemeria plants. Disease symptoms caused by this virus mainly consisted of yellow spots on the leaves based on which the name alstroemeria yellow spot virus (AYSV) was coined. A host range analysis was performed and a polyclonal antiserum was produced against purified AYSV ribonucleoproteins which only reacted with the homologous antigen and not with any other (established or tentative) orthotospovirus from a selection of American and Asian species. Upon thrips transmission assays the virus was successfully transmitted by a population of Thrips tabaci. The entire nucleotide sequence of the M and S RNA segments was elucidated by a conventional cloning and sequencing strategy, and contained 4797 respectively 2734 nucleotides (nt). Simultaneously, a next generation sequencing (NGS) approach (RNAseq) was employed and generated contigs covering the entire viral tripartite RNA genome. In addition to the M and S RNA nucleotide sequences, the L RNA (8865 nt) was obtained. The nucleocapsid (N) gene encoded by the S RNA of this virus consisted of 819 nucleotides with a deduced N protein of 272 amino acids and by comparative sequence alignments to other established orthotospovirus species showed highest homology (69.5% identity) to the N protein of polygonum ringspot virus. The data altogether support the proposal of AYSV as a new orthotospovirus species within a growing clade of orthotospoviruses that seem to share the Middle East basin as a region of origin.

Structure of the 30S ribosomal decoding complex at ambient temperature.

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Å resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA–protein complexes at near-physiological temperatures.

The influence of nucleotide sequence of RNA 5′ end on the electrostatic interaction energy of IFIT5 proteins with RNA

The influence of nucleotide sequence of RNA 5′ end on the electrostatic interaction energy of IFIT5 proteins with RNA

Complete Genome Sequences of Three Tomato Aspermy Virus Isolates in Japan.

ABSTRACTWe report here the first complete nucleotide sequences of genomic RNAs of three Tomato aspermy virus (TAV) isolates in Japan. Analysis of these sequences showed that they have unique characteristics in RNAs 2 and 3. The Japanese isolates are similar to each other compared to other TAVs.

Identification of a viroid-like RNA in a lychee Transcriptome Shotgun Assembly

In the course of a systematic screen of plant transcriptomic data, the sequence of a novel viroid-like RNA was identified in four contigs in a recent Transcriptome Shotgun Assembly (TSA) from lychee in China. Portions of this sequence are closely related to the central conserved region (CCR) and terminal conserved region (TCR) present in members of the genus Apscaviroid, sequences that are important criteria for viroid classification. RT-PCR with two divergent adjacent primers amplified the full 304 nucleotide sequence of this viroid-like RNA which can be folded into the rod-like secondary structure that is a typical feature of viroids in the family Pospiviroidae. Northern-blot hybridization following denaturing PAGE and sequential polyacrylamide gel electrophoresis (sPAGE) revealed the presence of both circular and linear forms of the viroid-like RNA in lychee leaf tissue. Phylogenetic analysis indicates that this RNA groups with known members of the genus Apscaviroid with a maximum pairwise sequence identity of <62%. Field surveys revealed the presence of this viroid-like RNA in most lychee samples collected in China; however, it was not found in the accessions in a germplasm collection of lychees that originated from a wide range of other countries. None of the four herbaceous hosts and one woody host tested were susceptible to infection with the lychee viroid-like RNA via agroinoculation and RNA inoculation. The provisional name lychee viroid-like RNA (LVd-like RNA) is proposed for this tentative new species in the genus Apscaviroid.

Identification of oral cavity biofilm forming bacteria and determination of their growth inhibition by Acacia arabica, Tamarix aphylla L. and Melia azedarach L. medicinal plants

ObjectiveBiofilms are complex, multi-species bacterial communities that colonize the oral cavity in the form of plaque and are known to cause dental caries and periodontal disease. Present study demonstrated the potential of three selected medicinal plants against isolated and identified dental biofilm forming strains. MethodsPathogenic bacteria from dental biofilms were isolated, cultured, identified by phylogenetic analysis using PCR-based 16S ribosomal RNA (or 16S rRNA) nucleotide sequences and were analyzed for their biofilm forming capability. The antimicrobial activity of the three important medicinal plant extracts (Acacia arabica, Tamarix aphylla L. and Melia azadirachta L.) was determined against the highest biofilm forming bacteria. ResultsPhylogenetic analysis revealed that the 19 strains belonged to Proteobacteria, Firmicutes and Actinobacteria. Among the 19 isolates, eleven strains were found to possess high biofilm formation capability comparatively and antimicrobial activity assay showed that the selected plants considerably inhibited their growth. Extract from A. arabica stem had strong effect on the ability of bacteria isolated from dental carries as evident by up to 73% reduction in biofilm formation on surface pre-treated with extract from this plant. The same extract also showed tremendous biofilm cleaning potential up to 87% of the biofilm. ConclusionThe results suggested that the extracts of selected medicinal plants could be used for protection against pathogenic dental biofilm causing bacteria and attempts should be taken by pharmaceutical industries to utilize it in dental caring products.

A new species of the genus Nicippe from Japan (Crustacea, Amphipoda, Pardaliscidae).

A new species of the pardaliscid amphipod, Nicippe recticaudata, from off Cape Toi, Japan, is named and described. This is the first record of Nicippe Bruzelius, 1859 from the western Pacific coast of the Japanese archipelago. Additionally, nucleotide sequences of nuclear 28S ribosomal RNA and histone H3 as well as mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA from the holotype and paratypes were determined. The morphological characteristics and the COI distance values enforced the distinctiveness of N. recticaudata sp. n. among the known Nicippe species. Nicippe recticaudata sp. n. closely resembles N. tumida Bruzelius, 1859 in having a two-dentate posterior margin of usoromite 1. However, the former is distinguished from the latter by the posterior margin of merus of pereopod 4 with 5–6 setae, anterior margin of merus of pereopod 5 with 9–10 setae, and telson with straight inner margin, tapering proximally. A key to the species of Nicippe is provided.

The complete genome sequence of a double-stranded RNA mycovirus from Fusarium graminearum strain HN1.

The complete nucleotide sequence of a double-stranded RNA (dsRNA) mycovirus, Fusarium graminearum dsRNA virus 5 (FgV5), was identified and characterized. The FgV5 genome comprises two dsRNA genome segments of 2030bp and 1740bp. FgV5 dsRNA1 contains a single open reading frame (ORF1), which is predicted to encode a protein of 613 amino acids (aa) with a molecular mass of 70.4kDa and has a conserved RNA-dependent RNA polymerase (RdRp) motif. FgV5 dsRNA2 is predicted to contain two discontinuous ORFs (ORF2 and ORF3) that code for products of unknown function. Sequence comparisons showed that FgV5 has the highest aa sequence identities to Fusarium graminearum virus 4 (FgV4) (83.01% for ORF1, 78.70% for ORF2, and 76.27% for ORF3), suggesting that FgV5 and FgV4 should be regarded as members of different species. Phylogenetic analysis indicated that FgV5 belongs to a taxonomically unassigned dsRNA mycovirus group that is related to the families Amalgaviridae and Partitiviridae. Here, we propose that FgV5 and related viruses are members of a yet to be named and formally recognized new family.

FIRST REPORT OF PEPPER CRYPTIC VIRUS 2 INFECTING PEPPER IN CHINA

Pepper cryptic virus 2 (PCV-2) is a double-stranded RNA virus which belongs to the genus Deltapartitivirus in the family Partitiviridae (Nibert et al., 2014; Sabanadzovic and Valverde, 2011). PCV-2 was previously reported infecting pepper in the USA and India (Saritha et al., 2016). In 2015, twenty pepper (Capsicum annuum L.) plants showing mosaic, crinkle and stunting symptoms were collected in Chongqing and used to detect potential viruses by high throughput sequencing using the Illumina HiSeq 2000 platform. Small RNA sequences between 18 and 26 nt in size were assembled and aligned. Five and three contigs were mapped to the double-stranded RNA1 and RNA2 of PCV-2 respectively. The primers PCV2-R1F (5’-AGAATTTTCCAAGCCGTTTACTT-3’)/ PCV2-R1R (5’-GATTAAGCTTCCAATTCAATGGT-3’) and PCV2-R2F (5’- AGAGCGGTGTGTTGGCAG-3’)/PCV2-R2R (5’-TAGTGCCTTATGCCCAAT-3’), designed in RNA-1 and RNA-2 of PCV-2 were used to confirm the presence of PCV-2 in pepper samples by RT-PCR. The partial nucleotide sequence of RNA1 (KX905077) and RNA2 (KX905078) obtained from PCV-2 isolate BB13 consisted of 1245 nt and 1487 nt, and shared highest identity of 97.1% and 99.8% with isolate HW-01 (JN117278) and ChS1 (KR676354), respectively. Nine out of the 20 samples that were tested by RT-PCR with specific primers HW-1/HW-1R (Sabanadzovic and Valverde, 2011) were positive for PCV-2. The high throughput sequencing data showed the occurrence of additional putative viruses such as Cucumber mosaic virus (CMV) in pepper. Therefore, symptoms could not be attributed only to PCV-2. To our knowledge, this is the first report of PCV-2 infecting pepper in China.

Identification of Quorum Quenching Bacteria and Its Biocontrol Potential Against Soft Rot Disease Bacteria, Dickeya dadantii

Dickeya dadantii is one of newly found bacteria causing soft rot on orchids in Indonesia. Infected plants showed severe rot rapidly only in few days. An effort to control the bacteria was conducted by utilizing selected quorum quenching (QQ) inducer bacteria which produce AHL-lactonase by aii A gene. The aims of this research were to screen and identify of quorum quenching bacteria, and also assayed their biocontrol potential ability against D. dadantii in laboratory. The screening of QQ bacteria was achieved using the anti-QS test, anti-microbial activity, and detection of aii A gene using specific primer . The determination of the ability against D. dadantii was done using the soft rot assay on potato and orchid . Among thirty one bacteria isolates screened, four isolates (in succession namely B37, BT2, GG3, and GG6) were selected to control D. dadantii . All of these bacteria showed QQ ability to suppress the virulence of D. dadantii infection on orchids, significantly. Based on nucleotide sequences of 16S ribosomal RNA, those of bacteria isolates had the highest identity with Brevibacillus brevis , Bacillus cereus ATCC14579, Bacillus cereus ATCC14579 and Bacillus thuringiensis ATCC 10792. Brevibacillus brevis was reported for the first time as QQ bacteria in this study.

Phylogeny of Egyptian isolates of Cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV) infecting Solanum lycopersicum

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.

Ribosome structural dynamics in translocation: yet another functional role for ribosomal RNA.

Ribosomes are remarkable ribonucleoprotein complexes that are responsible for protein synthesis in all forms of life. They polymerize polypeptide chains programmed by nucleotide sequences in messenger RNA in a mechanism mediated by transfer RNA. One of the most challenging problems in the ribosome field is to understand the mechanism of coupled translocation of mRNA and tRNA during the elongation phase of protein synthesis. In recent years, the results of structural, biophysical and biochemical studies have provided extensive evidence that translocation is based on the structural dynamics of the ribosome itself. Detailed structural analysis has shown that ribosome dynamics, like aminoacyl-tRNA selection and catalysis of peptide bond formation, is made possible by the properties of ribosomal RNA.

RNAdualPF: software to compute the dual partition function with sample applications in molecular evolution theory

BackgroundRNA inverse folding is the problem of finding one or more sequences that fold into a user-specified target structure s 0, i.e. whose minimum free energy secondary structure is identical to the target s 0. Here we consider the ensemble of all RNA sequences that have low free energy with respect to a given target s 0.ResultsWe introduce the program RNAdualPF, which computes the dual partition function Z ∗, defined as the sum of Boltzmann factors exp(−E(a,s 0)/RT) of all RNA nucleotide sequences a compatible with target structure s 0. Using RNAdualPF, we efficiently sample RNA sequences that approximately fold into s 0, where additionally the user can specify IUPAC sequence constraints at certain positions, and whether to include dangles (energy terms for stacked, single-stranded nucleotides). Moreover, since we also compute the dual partition function Z ∗(k) over all sequences having GC-content k, the user can require that all sampled sequences have a precise, specified GC-content.Using Z ∗, we compute the dual expected energy 〈E ∗〉, and use it to show that natural RNAs from the Rfam 12.0 database have higher minimum free energy than expected, thus suggesting that functional RNAs are under evolutionary pressure to be only marginally thermodynamically stable.We show that C. elegans precursor microRNA (pre-miRNA) is significantly non-robust with respect to mutations, by comparing the robustness of each wild type pre-miRNA sequence with 2000 [resp. 500] sequences of the same GC-content generated by RNAdualPF, which approximately [resp. exactly] fold into the wild type target structure. We confirm and strengthen earlier findings that precursor microRNAs and bacterial small noncoding RNAs display plasticity, a measure of structural diversity.ConclusionWe describe RNAdualPF, which rapidly computes the dual partition function Z ∗ and samples sequences having low energy with respect to a target structure, allowing sequence constraints and specified GC-content. Using different inverse folding software, another group had earlier shown that pre-miRNA is mutationally robust, even controlling for compositional bias. Our opposite conclusion suggests a cautionary note that computationally based insights into molecular evolution may heavily depend on the software used.C/C++-software for RNAdualPF is available at http://bioinformatics.bc.edu/clotelab/RNAdualPF.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1280-6) contains supplementary material, which is available to authorized users.

In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria.

Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes.

Association between targeted somatic mutation (TSM) signatures and HGS‐OvCa progression

Evidence already exists that the activation‐induced cytidine deaminase (AID/APOBEC) and the adenosine deaminase (ADAR) families of enzymes are implicated as powerful mutagens in oncogenic processes in many somatic tissues. Each deaminase is identified by the DNA or RNA nucleotide sequence (“motif”) surrounding the nucleotide targeted for deamination. The primary objective of this study is to develop an in silico approach to identify nucleotide sequence changes of the target motifs of key deaminases during oncogenesis. If successful, a secondary objective is to investigate if such changes are associated with disease progression indicators that include disease stage and progression‐free survival time. Using a discovery cohort of 194 high‐grade serous ovarian adenocarcinoma (HGS‐OvCa) exomes, the results confirm the ability of the novel in silico approach used to identify changes in the preferred target motifs for AID, APOBEC3G, APOBEC3B, and ADAR1 during oncogenesis. Using this approach, a set of new cancer‐progression associated signatures (C‐PASs) were identified. Furthermore, it was found that the C‐PAS identified can be used to differentiate between the cohort of patients that remained progression‐free for longer than 60 months, from those in which disease progressed within 60 months (sensitivity 95%, specificity 90%). The spectrum of outcomes observed here could provide a foundation for future clinical assessment of susceptibility variants in ovarian, and several other cancers as disease progresses. The ability of the in silico methodology used to identify changes in deaminase motifs during oncogenesis also suggests new links between immune system function and tumorigenesis.

SwiSpot: modeling riboswitches by spotting out switching sequences.

Riboswitches are cis-regulatory elements in mRNA, mostly found in Bacteria, which exhibit two main secondary structure conformations. Although one of them prevents the gene from being expressed, the other conformation allows its expression, and this switching process is typically driven by the presence of a specific ligand. Although there are a handful of known riboswitches, our knowledge in this field has been greatly limited due to our inability to identify their alternate structures from their sequences. Indeed, current methods are not able to predict the presence of the two functionally distinct conformations just from the knowledge of the plain RNA nucleotide sequence. Whether this would be possible, for which cases, and what prediction accuracy can be achieved, are currently open questions. Here we show that the two alternate secondary structures of riboswitches can be accurately predicted once the 'switching sequence' of the riboswitch has been properly identified. The proposed SwiSpot approach is capable of identifying the switching sequence inside a putative, complete riboswitch sequence, on the basis of pairing behaviors, which are evaluated on proper sets of configurations. Moreover, it is able to model the switching behavior of riboswitches whose generated ensemble covers both alternate configurations. Beyond structural predictions, the approach can also be paired to homology-based riboswitch searches. SwiSpot software, along with the reference dataset files, is available at: http://www.iet.unipi.it/a.bechini/swispot/Supplementary information: Supplementary data are available at Bioinformatics online. a.bechini@ing.unipi.it.

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15% of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Why does biological evolution use genetic algorithms?

Why does biological evolution use genetic algorithms?