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PubMed1992

Direct sequence analysis of amplified dengue virus genomic RNA from cultured cells, mosquitoes and mouse brain.

A method is described for direct sequence analysis of selected regions of dengue virus genomic RNA in infected tissues. Using specific primers, total high-molecular-weight infected-cell RNA is reverse transcribed to single-stranded (ss) complementary DNA, amplified using the polymerase chain reaction (PCR) and sequenced using ssDNA obtained after lambda exonuclease digestion of one strand of the PCR product (R.G. Higuchi and H. Ochman, Nucleic Acids Research, 17, 5865, 1989). Sequence data for the envelope protein gene of two dengue-3 virus isolates were obtained using RNA from small numbers (10(5)) of cultured mosquito or monkey kidney cells, from one mg of infected mouse brain and from 1/300th of an infected Toxorhynchites amboinensis mosquito. Independent determinations showed that errors occurring during reverse transcription or PCR were not represented to a significant degree in the sequence of the amplified DNA. The method does not depend on extensive passaging of virus or large-scale growth to generate material for sequencing and therefore provides a means of obtaining sequence data for unadapted dengue virus isolates.

Aedes↗
PubMed2001

[Nuclear ribosomal RNA small subunit (18S rRNA) nucleotide sequencing and characterization of sailonggu(whole bone of Myospalax baileyi Thomas)].

OBJECTIVE: Sequencing the nuclear ribosomal RNA small subunit (18S rRNA) gene of Myospalax baileyi (Cricetidae) to develop an ultimate and definitive means for origin identification of genuine Sailonggu. METHODS: The total DNA was prepared from dried tail tissues. The nuclear 18S rRNA gene region was amplified by PCR using a consensus primer set and its nucleotide sequence was determined by PCR direct sequencing. The characteristic analysis of 18S rRNA sequences was generated using software program Genetyx-SV/R Version 10.1. RESULTS: The entire 18S rRNA gene region of M. baileyi spanded 1851 bp in length. Although multiple alignment of sequence indicates that there are only lower homology (72.04%-72.18%) comparing with its two alias Mus musculus (GenBank Accession number X00686) and Rattus norvegicus (M11188) (Muridae), their highly conservative domain is located in 1020-1509 nt. There are many variable sites from upstream of 5'-end, which could provide a novel information for molecular recognition of Sailonggu. CONCLUSION: DNA sequencing could be a useful and reliable tool in the origin identification of genuine Sailonggu.

Animals↗
PubMed1987

Nonuniformity of nucleotide substitution rates in molecular evolution: computer simulation and analysis of 5S ribosomal RNA sequences.

The effects of temporal (among different branches of a phylogeny) and spatial (among different nucleotide sites within a gene) nonuniformities of nucleotide substitution rates on the construction of phylogenetic trees from nucleotide sequences are addressed. Spatial nonuniformity may be estimated by using Shannon's (1948) entropy formula to measure the Relative Nucleotide Variability (RNV) at each nucleotide site in an aligned set of sequences; this is demonstrated by a comparative analysis of 5S rRNAs. New methods of constructing phylogenetic trees are proposed that augment the Unweighted Pair-Group Using Arithmetic Averages (UPGMA) algorithm by estimating and compensating for both spatial and temporal nonuniformity in substitution rates. These methods are evaluated by computer simulations of 5S rRNA evolution that include both kinds of nonuniformities. It was found that the proposed Reference Ratio Method improved both the ability to reconstruct the correct topology of a tree and also the estimation of branch lengths as compared to UPGMA. A previous method (Farris et al. 1970; Klotz et al. 1979; Li 1981) was found to be less successful in reconstructing topologies when there is high probability of multiple mutations at some sites. Phylogenetic analyses of 5S rRNA sequences support the endosymbiotic origins of both chloroplasts and mitochondria, even though the latter exhibit an accelerated rate of nucleotide substitution. Phylogenetic trees also reveal an adaptive radiation within the eubacteria and another within the eukaryotes for the origins of most major phyla within each group during the Precambrian era.

Animals↗
PubMed1992

Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites.

The sequence of the most variable part of the small subunit ribosomal RNA (SSU rRNA) gene, comprising 800 bases, was analysed for 9 Leishmania taxa and compared with those of Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. Considerable differences were observed between the sequence of the Leishmania taxa on the one hand and those of Crithidia and Trypanosoma on the other. Amongst the Leishmania taxa only a few point mutations were found, all located within 2 sequence blocks in the central part of the SSU rRNA gene, which are unique for Kinetoplastida. These unique sequences were used for the development of kinetoplastid-specific probes and a Leishmania-specific PCR assay of high sensitivity (less than 10 parasites could be detected). Based on the observed point-mutations an identification of the Leishmania parasites, according to complex, could be achieved by direct sequencing, restriction fragment analysis or single-stranded conformation polymorphism of the PCR-generated fragments.

Animals↗
PubMed2005

Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome.

The organization of eukaryotic genomes into distinct structural and functional domains is important for the regulation and transduction of genetic information. Here, we investigated heterochromatin and euchromatin profiles of the entire fission yeast genome and explored the role of RNA interference (RNAi) in genome organization. Histone H3 methylated at Lys4, which defines euchromatin, was not only distributed across most of the chromosomal landscape but was also present at the centromere core, the site of kinetochore assembly. In contrast, histone H3 methylated at Lys9 and its interacting protein Swi6/HP1, which define heterochromatin, coated extended domains associated with a variety of repeat elements and small islands corresponding to meiotic genes. Notably, RNAi components were distributed throughout all these heterochromatin domains, and their localization depended on Clr4/Suv39h histone methyltransferase. Sequencing of small interfering RNAs (siRNAs) associated with the RITS RNAi effector complex identified hot spots of siRNAs, which mapped to a diverse array of elements in these RNAi-heterochromatin domains. We found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs. Our analyses also uncover an important role for the RNAi machinery in maintaining genomic integrity.

DNA Methylation↗
PubMed1995

Sequence analysis of the 5' untranslated region of pestivirus RNA demonstrated in interferons for human use.

We examined 46 samples of human interferons for the presence of pestivirus RNA by the reverse transcription-polymerase chain reaction, and found 30.4% were contaminated with pestivirus RNA. Comparative analysis of nucleotide sequences in the 5' untranslated region (UTR) of the pestivirus RNA detected from the interferons indicated that the contaminants are derived from bovine viral diarrhea virus, and that there are at least three genotypes, I, II and III, among them. The incidence of genotypes I, II and III was 86%, 7% and 7%, respectively. Most of the nucleotide changes at variable regions in the 5' UTR were covariant, with complementary substitutions at other positions of the stem regions in secondary structures.

Base Sequence↗
PubMed2004

The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4.

We report the complete genome sequence of Zymomonas mobilis ZM4 (ATCC31821), an ethanologenic microorganism of interest for the production of fuel ethanol. The genome consists of 2,056,416 base pairs forming a circular chromosome with 1,998 open reading frames (ORFs) and three ribosomal RNA transcription units. The genome lacks recognizable genes for 6-phosphofructokinase, an essential enzyme in the Embden-Meyerhof-Parnas pathway, and for two enzymes in the tricarboxylic acid cycle, the 2-oxoglutarate dehydrogenase complex and malate dehydrogenase, so glucose can be metabolized only by the Entner-Doudoroff pathway. Whole genome microarrays were used for genomic comparisons with the Z. mobilis type strain ZM1 (ATCC10988) revealing that 54 ORFs predicted to encode for transport and secretory proteins, transcriptional regulators and oxidoreductase in the ZM4 strain were absent from ZM1. Most of these ORFs were also found to be actively transcribed in association with ethanol production by ZM4.

Biological Transport↗
PubMed1991

Sequence analysis of the S RNA of the African arenavirus Mopeia: an unusual secondary structure feature in the intergenic region.

Mopeia virus is an apparently nonpathogenic African arenavirus which can protect animals from subsequent challenge by the closely related Lassa virus. As a step toward understanding these differences in pathogenicity and the means by which Mopeia virus infection can protect against subsequent Lassa virus infection, cDNA clones corresponding to 3419 nucleotides of Mopeia virus S RNA were isolated and sequenced. Two open reading frames, encoding the glycoprotein precursor (GPC) and nucleocapsid (N) proteins, were located in the ambisense arrangement characteristic of the arenaviruses. Comparison of the amino acid sequences of the translation products with those of two Lassa virus strains showed considerable conservation, with 74 and 80% identity for the two glycoproteins G1 and G2, and 74% identity for the N protein. The putative dibasic site of GPC cleavage (R-R) was conserved, as were the potential N-linked glycosylation sites. A striking difference between Mopeia virus and Lassa virus was identified in the noncoding intergenic region. Instead of the single hairpin structure formed by base-pairing of complementary sequences which is usually found, the Mopeia virus S RNA has the potential to form two hairpins. These hairpins were similar in sequence and may have been formed in a duplication event during RNA replication. The possible contribution of this secondary structure feature to differences in pathogenicity between Mopeia and Lassa viruses is discussed.

Amino Acid Sequence↗
PubMed2025

A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts.

Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.

RNA Editing↗
PubMed2002

Expression profile and genome location of cDNA clones from an infant human trabecular meshwork cell library.

PURPOSE: To delineate the profile of genes expressed in infant human trabecular meshwork and identify candidate genes for glaucoma. METHODS: Human trabecular meshwork cell cultures were established from six young donors. A cDNA library was made from the combined trabecular meshwork mRNA. The end-sequence of random clones was determined by direct sequencing. These sequences were then analyzed by a National Center for Biotechnology Information (NCBI, Bethesda, MD) database search. Nucleotide searches were performed using the BLASTN (ver. 2.1.3; against the nonredundant nucleic acid sequence) and dbEST databases (both provided by NCBI in the public domain at www.ncbi.nlm.nih.gov). RESULTS: Sequences from 1118 clones from this nonamplified trabecular meshwork cDNA library were categorized. Of these, 877 expressed sequence tags (ESTs) (78.7%) were known genes. One hundred thirty-nine ESTs (12.5%) showed close identity to EST sequences reported in the public domain database (dbEST). Thirteen ESTs (1.2%) showed no significant similarity to known genes or ESTs in the public databases and were thus defined as novel ESTs. The most abundant genes expressed by the human trabecular meshwork included ferritin H, eukaryotic translation elongation factor 1-alpha, ferritin L, fibronectin, and TIMP-1. Ferritin H was the most abundant transcript, making up more than 4% of the genes expressed by the human trabecular meshwork. Extracellular matrix proteins were also highly expressed. The chromosome location of the trabecular meshwork ESTs is reported. CONCLUSIONS: A profile of genes expressed by human trabecular meshwork is presented. Thirteen novel ESTs were identified. The combined information obtained from expression analysis and chromosomal localization of trabecular meshwork cDNAs should be valuable in identifying candidate genes for glaucoma.

Cells, Cultured↗
PubMed2000

Phylogenetic relationships of Acanthocephala based on analysis of 18S ribosomal RNA gene sequences.

Acanthocephala (thorny-headed worms) is a phylum of endoparasites of vertebrates and arthropods, included among the most phylogenetically basal tripoblastic pseudocoelomates. The phylum is divided into three classes: Archiacanthocephala, Palaeacanthocephala, and Eoacanthocephala. These classes are distinguished by morphological characters such as location of lacunar canals, persistence of ligament sacs in females, number and type of cement glands in males, number and size of proboscis hooks, host taxonomy, and ecology. To understand better the phylogenetic relationships within Acanthocephala, and between Acanthocephala and Rotifera, we sequenced the nearly complete 18S rRNA genes of nine species from the three classes of Acanthocephala and four species of Rotifera from the classes Bdelloidea and Monogononta. Phylogenetic relationships were inferred by maximum-likelihood analyses of these new sequences and others previously determined. The analyses showed that Acanthocephala is the sister group to a clade including Eoacanthocephala and Palaeacanthocephala. Archiacanthocephala exhibited a slower rate of evolution at the nucleotide level, as evidenced by shorter branch lengths for the group. We found statistically significant support for the monophyly of Rotifera, represented in our analysis by species from the clade Eurotatoria, which includes the classes Bdelloidea and Monogononta. Eurotatoria also appears as the sister group to Acanthocephala.

Acanthocephala↗
PubMed1997

A helix 1-extended hairpin ribozyme exhibits altered cleavage behavior in vitro.

The catalytic domain of a hairpin ribozyme was incorporated at the 3'-end of a 254-base antisense RNA directed against the RNA of human immunodeficiency virus type 1 (HIV-1), generating a hairpin ribozyme with a largely extended helix 1. In parallel, a catalytic antisense RNA based on a hammerhead ribozyme was directed toward the same cleavage motif in the HIV-1 target. Both ribozymes were expected to create identical cleavage products. Cleavage analysis in vitro confirmed that the hammerhead ribozyme delivered the expected cleavage products. However, the helix 1-extended hairpin ribozyme catalyzed additional RNA cleavage at several unexpected sites, which were mapped. Some of the 3' cleavage products had other nucleotides than G at their 5'-terminus, indicating that the helix 1-extended hairpin ribozyme was able to cleave bonds other than NpG+1. Inspection of the sequence context of the different cleavage sites suggested that unconventional helices 2 in combination with an asymmetric loop A consisting of up to 32 unpaired nucleotides in the substrate strand were formed. A second variant of a helix 1-extended hairpin ribozyme that differed in two nucleotides gave consistent results.

HIV-1↗
PubMed2006

Structural and evolutionary classification of G/U wobble basepairs in the ribosome.

We present a comprehensive structural, evolutionary and molecular dynamics (MD) study of the G/U wobble basepairs in the ribosome based on high-resolution crystal structures, including the recent Escherichia coli structure. These basepairs are classified according to their tertiary interactions, and sequence conservation at their positions is determined. G/U basepairs participating in tertiary interactions are more conserved than those lacking any interactions. Specific interactions occurring in the G/U shallow groove pocket--like packing interactions (P-interactions) and some phosphate backbone interactions (phosphate-in-pocket interactions)--lead to higher G/U conservation than others. Two salient cases of unique phylogenetic compensation are discovered. First, a P-interaction is conserved through a series of compensatory mutations involving all four participating nucleotides to preserve or restore the G/U in the optimal orientation. Second, a G/U basepair forming a P-interaction and another one forming a phosphate-in-pocket interaction are replaced by GNRA loops that maintain similar tertiary contacts. MD simulations were carried out on eight P-interactions. The specific GU/CG signature of this interaction observed in structure and sequence analysis was rationalized, and can now be used for improving sequence alignments.

Base Pairing↗
PubMed1989

Nucleotide sequence analysis of the small (S) RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae.

The nucleotide sequence of the small (S) RNA segment of the Bunyamwera virus genome has been determined. The S RNA is 961 bases in length and, in common with other bunyaviruses, encodes two proteins, N and NSs, in overlapping reading frames. A six-way alignment of the amino acid sequences of the N and NSs proteins of viruses representing three serogroups within the Bunyavirus genus indicates regions which are strongly conserved, and provides targets for future analysis of protein function.

Amino Acid Sequence↗
PubMed1999

Confirmation of predicted edits and demonstration of unpredicted edits in Acanthamoeba castellanii mitochondrial tRNAs.

In the amoeboid protozoon Acanthamoeba castellanii 13 of the 16 mtDNA-encoded tRNA sequences have mis-matches at one or more of the first three positions in the acceptor stem. A previous study had indicated that these mis-matches are corrected by a form of RNA editing. In the present study, the pattern of editing was further investigated by sequence analysis of both halves of the acceptor stem of 11 mtDNA-encoded tRNAs. The results confirm all of the remaining editing sites predicted on the basis of the secondary structure modelling of A. castellanii mitochondrial tRNAs, and identify two unexpected edits. We also investigated the expression and editing of transcripts of an unusual trnX gene specifying an eight-nucleotide anticodon loop sequence. Although no mature 3'-CCAOH-containing tRNAX products were detected, editing was observed in some circularized tRNAX clones. The implications of these results with respect to the mechanism of editing and the evolutionary origin of this process are discussed.

Acanthamoeba↗
PubMed1996

Sensitive detection of human Caliciviridae by RT-PCR.

A semi-nested reverse transcriptase-polymerase chain reaction (RT-PCR) was developed for the detection of human Caliciviridae. The method was evaluated on faecal samples from patients with gastroenteritis sent to the Norwegian National Institute of Public Health for routine diagnosis by direct electron microscopy (EM). Of 166 samples, 49 were found to contain Caliciviridae by EM, while 7 samples contained other viruses. A total of 74 samples was positive by PCR, including all the samples with EM detectable Caliciviridae, while specimens containing other agents were negative. Phylogenetic analysis of RNA sequences from 14 Norwegian samples indicated that the viruses present in Norway are evenly distributed when compared to sequences of human Caliciviridae from other countries. The PCR primers should therefore be useful for samples from other regions. The phylogenetic analysis did not cluster viruses with a calici-like morphology, but mingled them with sequences from Norwalk-like viruses, indicating that the two morphological types do not represent separate genogroups.

Base Sequence↗
PubMed2001

A complex human immunodeficiency virus type 1 A/G/J recombinant virus isolated from a seronegative patient with AIDS from Benin, West Africa.

A human immunodeficiency virus type 1 (HIV-1(B76)) originating from Benin (West Africa) was isolated and characterized. The patient had severe clinical AIDS and presented an unusual serological profile. Only one out of five different detection assays was able to demonstrate the presence of antibodies to HIV, whereas confirmatory assays remained indeterminate. In contrast, both plasma viral load and p24 antigen level were unusually high. HIV-1 infection was proved by viral RNA and proviral DNA amplification. HIV-1(B76) partially purified lysate reacted strongly with all anti-HIV-1-positive sera from the region but B76 plasma did not react with subtype A control viral antigen. This patient is likely to have had severe acquired immune dysfunction explaining her lack of immunological reactivity. Phylogenetic analysis of the genome identified a complex HIV-1 A/G/J recombinant. The gag and pol genes, and the majority of nef,are characteristic of subtype A; the gag/pol junction, the 3' end of pol, vpu and env genes were characteristic of subtype G; vif, vpr and the 5' end of nef were subtype J. In addition, part of the HIV-1(B76) genome had considerable sequence similarity with the previously described CRF06 cpx (BFP90) isolate. HIV-1(B76) did not exhibit any remarkable replication properties or cell tropism in vitro.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples.

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.

Ammonia↗