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At least 649 records · Page 36Linked to original sources
PubMed2002

Detection and analysis of hairpin II, an essential metastable structural element in viroid replication intermediates.

In (-)-stranded replication intermediates of the potato spindle tuber viroid (PSTVd) a thermodynamically metastable structure containing a specific hairpin structure (HP II) has been proposed to be essential for viroid replication. In the present work a method was devised allowing the direct detection of the HP II structure in vitro and in vivo using a biophysical approach. An RNA oligonucleotide was constructed which specifically binds to the HP II loop region in transient (-)-strand intermediates. Analysis of the resulting oligonucleotide/HP II complexes on temperature-gradient gels enabled us to follow the formation of HP II during in vitro transcription by T7 RNA polymerase. Moreover, we were able to demonstrate the formation of HP II during viroid replication in potato (Solanum tuberosum) cells.

Base Sequence↗
PubMed1993

Generation and selection of coronavirus defective interfering RNA with large open reading frame by RNA recombination and possible editing.

All of the coronavirus defective interfering (DI) RNAs analyzed thus far contain an open reading frame (ORF) from which DI RNA-specific protein(s) are translated, although the function of the DI-specific protein and the significance of the ORF are not known. A complete cDNA clone of a mouse hepatitis virus (MHV) DI RNA, NE-1, containing a single nucleotide deletion in the 5' region of the ORF was obtained and analyzed. Due to this single nucleotide deletion, a DI-specific protein of 7.5-kDa was made from NE-1, in contrast to the 88-kDa protein made from the wild-type DI RNA. NE-1 RNA was efficiently replicated after transfection into MHV-infected cells. However, after one passage of NE-1 RNA-containing virus, the 88-kDa wild-type protein was synthesized, indicating that the large ORF was restored during NE-1 DI RNA replication. Sequence analysis of NE-1 DI RNA from infected cells demonstrated that in approximately half of the DI RNA population, the ORF was restored by RNA recombination between NE-1 DI RNA and helper virus genomic sequence. The sequences of other DI RNAs contained an additional nontemplated A at the five-A sequence nine nucleotides upstream of the deletion site, resulting in a stretch of six consecutive As. In these "edited"-type DI RNAs, the original nucleotide deletion was maintained and no RNA recombination was observed. This "editing" produced an ORF of the same size as the wild-type DI RNA. We conclude that the DI RNA with a large ORF has a selective advantage. There was no significant difference in replication efficiency among these RNAs when they replicated alone. However, cotransfection of two DI RNA species and time course experiments suggested that homologous interference and other mechanism(s) during the early stage of virus multiplication are responsible for the accumulation of DI RNAs containing the large ORF.

Amino Acid Sequence↗
PubMed2000

Phylogenetic analysis of the 5' subterminal region of isolates of Leishmania RNA virus-1.

Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus present in some Leishmania species. The virus genome consists of a 450-nucleotide, 5' untranslated region (UTR) followed by the coat gene and the RNA-dependent RNA polymerase (RDRP). It has been shown that the 5' end UTR of the genome promotes internal initiation of translation in an in-vitro assay, indicating the presence of an internal ribosomal entry site (IRES) element upstream of the coat gene. The nucleotide sequences of the 5' subterminal regions of six new isolates of LRV1, of different geographical origins, have now been determined. The RNA folding of the 5' subterminal region of LRV1 has been predicted, using a combination of thermodynamic parameters and folding constraints based on nucleotide substitutions. Furthermore, a putative pyrimidine-rich region (a feature unique to all IRES elements), which is complementary to the Leishmania 18S rRNA, has been identified. The significance and relevance of these findings in the context of the function of the 5' UTR of LRV1 as an IRES element are discussed.

Animals↗
PubMed2004

Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs.

We present an EST sequence assembler that specializes in reconstruction of pristine mRNA transcripts, while at the same time detecting and classifying single nucleotide polymorphisms (SNPs) occuring in different variations thereof. The assembler uses iterative multipass strategies centered on high-confidence regions within sequences and has a fallback strategy for using low-confidence regions when needed. It features special functions to assemble high numbers of highly similar sequences without prior masking, an automatic editor that edits and analyzes alignments by inspecting the underlying traces, and detection and classification of sequence properties like SNPs with a high specificity and a sensitivity down to one mutation per sequence. In addition, it includes possibilities to use incorrectly preprocessed sequences, routines to make use of additional sequencing information such as base-error probabilities, template insert sizes, strain information, etc., and functions to detect and resolve possible misassemblies. The assembler is routinely used for such various tasks as mutation detection in different cell types, similarity analysis of transcripts between organisms, and pristine assembly of sequences from various sources for oligo design in clinical microarray experiments.

Animals↗
PubMed2005

RNA tertiary structure determination: NOE pathways construction by tabu search.

MOTIVATION: Liquid state nuclear magnetic resonance (NMR) spectroscopy has now been well established as a method for RNA tertiary structure determination. Most of the steps involved in the determination of RNA molecules are performed using computer programs. They however, do not apply to resonance assignment being the starting point of the whole procedure. We propose a tabu search algorithm as a tool for automating this step. Nuclear overhause effect (NOE) pathway, which determines the assignment, is constructed during an analysis of possible connections between resonances within aromatic/anomeric region of two-dimensional NOESY spectrum resulting from appropriate NMR experiment. RESULTS: Computational tests demonstrate the superior performance of the tabu search algorithm as compared with the exact enumerative approach and genetic procedure applied to the experimental and simulated spectral data for RNA molecules. AVAILABILITY: The software package can be obtained upon request from Marta Szachniuk.

Algorithms↗
PubMed2000

Intestinal inflammatory pseudotumour with regional lymph node involvement: identification of a new bacterium as the aetiological agent.

Inflammatory pseudotumours are the morphological expression of diverse processes such as reactive/reparative, infective, and neoplastic. This paper reports an example of intestinal inflammatory pseudotumour, with identification of a newly characterized bacterium in the lesion. The patient presented with intestinal obstruction. Laparotomy revealed a tumour in the terminal ileum causing stricture, and multiple enlarged regional lymph nodes. Histologically, the tumour and lymph nodes were composed of plump spindle cells disposed in a vague storiform pattern, and associated with lymphocytes and plasma cells. Immunohistochemical studies showed that most of the spindle cells were histiocytes (CD68 positive), prompting a search for a bacterial aetiology, akin to mycobacterial spindle cell pseudotumour. All histochemical stains for micro-organisms were unrewarding. Ultrastructural studies, however, revealed abundant bacteria within the spindle histiocytes. Polymerase chain reaction, using conserved oligonucleotide primers complementary to the 16S rRNA genes of eubacteria, was employed to amplify 16S rRNA gene fragments directly from the involved lymph node tissue. Phylogenetic analysis of the amplified DNA sequences revealed an organism with 99% sequence conformity to Pseudomonas veronii, a bacterium which has hitherto not been implicated in human infection. The importance of searching for an infective agent in inflammatory pseudotumour in the appropriate setting is re-emphasized.

Granuloma, Plasma Cell↗
PubMed2004

FAN: fingerprint analysis of nucleotide sequences.

FAN is a server for fingerprint analysis of nucleotide sequences. The server performs a search of submitted nucleotide sequences against the PRINTS database. Searches are performed directly against fingerprints using a codon position specific score matrix (PSSM) approach. The advantages of this approach are increased specificity for coding sequence (CDS) over non-CDS, and increased tolerance to base-substituting and frameshifting sequence errors. Furthermore, there is no need for prior translation of the nucleotide sequences. A web-based interface to the software is available at http://bioinf.man.ac.uk/cgi-bin/neil/ntfront.pl.

Amino Acid Motifs↗
PubMed2000

Abnormal T-cell repertoire is consistent with immune process underlying the pathogenesis of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC). Somatic mutations in the PIG-A gene result in the deficiency of several glycosylphosphatidylinositol-linked proteins from the surface of blood cells. This explains intravascular hemolysis but does not explain the mechanism of bone marrow failure that is almost invariably seen in PNH. In view of the close relationship between PNH and idiopathic aplastic anemia (IAA), it has been suggested that the 2 disorders might have a similar cellular pathogenesis, namely, that autoreactive T-cell clones are targeting HSCs. In this paper, we searched for abnormally expanded T-cell clones by size analysis of the complementarity-determining region 3 (CDR3) in the beta variable chain (BV) messenger RNA (mRNA) of the T-cell receptor (TCR) in 19 patients with PNH, in 7 multitransfused patients with hemoglobinopathy. and in 11 age-matched healthy individuals. We found a significantly higher degree of skewness in the TCR BV repertoire of patients with PNH, compared with controls (R(2) values 0.82 vs 0.91, P <.001). The mean frequency of skewed families per individual was increased by more than 2-fold in patients with PNH, compared with controls (28% +/- 19.6% vs 11.4% +/- 6%, P =.002). In addition, several TCR BV families were significantly more frequently skewed in patients with PNH than in controls. These findings provide experimental support for the concept that PNH, like IAA, has an immune pathogenesis. In addition, the identification of expanded T-cell clones by CDR3 size analysis will help to investigate the effect of HSC-specific T cells on normal and PNH HSCs.

Adolescent↗
PubMed2005

A class of 2D graphical representations of RNA secondary structures and the analysis of similarity based on them.

Based on the concepts of cell and system of graphical representation, a class of 2D graphical representations of RNA secondary structures are given in terms of classifications of bases of nucleic acids. The representations can completely avoid loss of information associated with crossing and overlapping of the corresponding curve. As an application, we make quantitative comparisons for a set of RNA secondary structures at the 3'-terminus of different viruses based on the graphical representations. The examination of similarities/dissimilarities illustrates the utility of the approach.

Algorithms↗
PubMed2000

[Analysis of hepatitis G virus genotypes in some areas of China].

OBJECTIVE: To study the genotype distribution of hepatitis G virus (HGV) isolated from different population in some areas of China. METHODS: A total of 12 strains of HGV were isolated from different areas of China (three from Guangxi Zhuang Autonomous Region, three from Hebei Province, one from Heilongjiang, Jiangxi, Guangdong, Jiangsu, Anhui, Jilin Provinces and Xinjiang Autonomous Region each). The HGV 5' untranslated region (UTR) of the 12 isolates was sequenced and compared with other 26 isolates previously reported (17 from China and nine from other countries). RESULTS: The nucleotide sequences of five HGV clones isolated from the same patient with HGV infection were identical, with the homology of 99.1% - 100%, indicating that variation of HGV in the same human body was very limited. Genotyping could be based on the sequence of one positive clone isolated from a patient with HGV infection. The homology of the nucleotide sequences in the 12 isolates was 96.8% - 99.4%, suggesting that they belonged to the same genotype. The homology of the 12 isolates was 88.0% - 89.7%, 87.2% - 90.3% and 94.7% - 96.3%, respectively, as compared with HGV genotypes 1 (U36380), 2 (U44402) and 3 (D90601), which indicated that all the 12 HGV isolates belonged to genotype 3. Phylogenetic distance analysis of the 38 isolates suggested that HGV strains isolated from China could be further divided into different subgroups. CONCLUSION: The majority of Chinese HGV isolates belonged to genotype 3 and they could be further divided into different subgroups.

Base Sequence↗
PubMed1996

A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences.

Analysis of restriction fragment length polymorphism of bacterial small-subunit (SSU) rRNA sequences represents a potential means for characterizing complex bacterial populations such as those found in natural environments. In order to estimate the resolution potential of this approach, we have examined the SSU rRNA sequences in the Ribosomal Database Project bank using a computer algorithm which simulates hybridization between DNA sequences. Simulated hybridizations between a primer or probe sequence and an SSU rRNA sequence yield a value for each potential hybridization. This algorithm has been used to evaluate sites for PCR primers and hybridization probes used for classifying SSU rRNA sequences. Our analysis indicates that length variation in terminal restriction fragments of PCR products from the SSU rRNA sequences can identify a wide spectrum of bacteria. We also observe that the majority of restriction fragment length variation is the result of insertions and deletions rather than restriction site polymorphisms. This approach is also used to evaluate the relative efficiency and specificity of a number of published hybridization probes.

Bacteria↗
PubMed2005

Epidemiology and genotypes of HEV in Wuhan.

BACKGROUND: Understanding the genotype and clinical features of the hepatitis E virus (HEV) are important for understanding its characteristics, for evaluating region-specific diagnostic assays, and producing vaccines. OBJECTIVES: To investigate the epidemiology and the genotypes of HEV among outpatients and inpatients in the Department of Infectious Diseases of Tongji Hospital in Wuhan, China. METHODS: Clinical data were elicited from the hospital records of patients who were clinically diagnosed with acute hepatitis between January 2000 and August 2004 (4920 patients). Of these cases, 120 patients with anti-HEV-IgM, IgG-positive were selected to analysis. Conserved genomic sequences of open reading frame 2 (345 bp) in the HEV gene were detected using polymerase chain reaction, 25 of which were cloned and sequenced. Clustal X and Mega software were used for phylogenetic analysis of genotypes strains. RESULTS: The HEV infection rate is gradually increasing in Wuhan. The number of male patients was 3.3-fold greater than the number of female patients found in clinical investigations. People aged 30-59 years are more susceptible to infection, and people are more susceptible in March-June. Twenty-five isolates shared the same genotype, genotype IV, with 82.61-98.55% nucleotide identity. This genotype had 76.52-81.74%, 70.43-73.04%, 76.52-81.16%, and 84.35-88.70% homology with the nucleotide sequence of HEV genotypes I-IV, respectively. Phylogenetic analysis suggested that these 25 isolates represented at least three different subtypes, but there were no significant differences found in the epidemiological features or liver function of patients with the three subtypes. CONCLUSIONS: HEV sequences isolated from patients in Wuhan belong to different subtypes of HEV genotype IV.

Adolescent↗
PubMed1997

Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines.

Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures of the swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.

Animals↗
PubMed2006

Assessment of arbuscular mycorrhizal fungal diversity in roots of Solidago gigantea growing in a polluted soil in Northern Italy.

The arbuscular mycorrhizal (AM) status of Solidago gigantea was investigated in a contaminated site of Northern Italy, where the chemical industry ACNA (Associated National Chemical Companies) was active till 1999. To counteract the devastating effects of chemicals and to allow re-vegetation, soil from an uncontaminated area was used to cover the highly polluted hills of the industrial site about 25 years ago. On the basis of the current floristic features, the hill was divided into four areas. Heavy metal content in soil and in plant shoots and roots was determined by chemical analysis. The AM fungal community colonizing S. gigantea was investigated from a morphological and a molecular point of view. All plants were modestly colonized, but the fungal structures within the roots were normal. By PCR-RFLP and sequencing of 18S rDNA, 14 AM fungal types were identified: three of them were present in all the considered areas and nine appeared to be specific to certain areas. Glomus was the predominant AM genus. Our analysis demonstrates the presence and the relatively high level of AM species variety and shows how a remediation programme based on cover-soil has been efficient to restore a community of AM fungi, tolerant enough to proliferate in a still contaminated soil.

Environmental Pollution↗
PubMed1985

Nucleotide sequence and genome organization of carnation mottle virus RNA.

The complete nucleotide sequence of carnation mottle genomic RNA (4003 nucleotides) is presented. The sequence was determined for cloned cDNA copies of viral RNA containing over 99% of the sequence and was completed by direct sequence analysis of RNA and cDNA transcripts. The sequence contains two long open reading frames which together can account for observed translation products. One translation product would arise by suppression of an amber termination codon and the sequence raises the possibility that a second suppression event could also occur. Sequence homology exists between a portion of the carnation mottle virus sequence and that of putative RNA polymerases from other RNA viruses.

Amino Acid Sequence↗
PubMed2006

The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data.

Substantial new features have been implemented at the Ribosomal Database Project in response to the increased importance of high-throughput rRNA sequence analysis in microbial ecology and related disciplines. The most important changes include quality analysis, including chimera detection, for all available rRNA sequences and the introduction of myRDP Space, a new web component designed to help researchers place their own data in context with the RDP's data. In addition, new video tutorials describe how to use RDP features. Details about RDP data and analytical functions can be found at the RDP-II website (http://rdp.cme.msu.edu/).

Databases, Nucleic Acid↗
PubMed2004

Analysis of RING finger genes required for embryogenesis in C. elegans.

The RING finger motif exists in E3 ligases of the ubiquitination pathway. These ubiquitin ligases bind to target proteins, leading to their modification by covalent addition of ubiquitin peptides. Current databases contain hundreds of proteins with RING finger motifs. This study investigates the role of RING finger genes in embryogenesis of the nematode, Caenorhabditis elegans. We expand the previous list of RING finger-containing genes and show that there are 103 RING finger-containing genes in the C. elegans genome. DNA microarrays of these 103 genes were probed with various RNA samples to identify 16 RING finger genes whose expression is enriched in the germline. RNA interference (RNAi) analysis was then used to determine the developmental role of these genes. One RING finger gene, C32D5.10, showed a dramatic larval arrest upon RNAi. Three RING finger genes exhibited embryonic lethality after RNAi. These three genes include par-2, and two small RING finger proteins: F35G12.9 (an ortholog of APC11) and ZK287.5 (an ortholog of rbx1). Embryos from RNAi of the APC11 and rbx1 orthologs were arrested in the cell cycle, confirming the role of these particular RING finger proteins in regulation of the cell cycle. genesis 38:1-12, 2004.

Amino Acid Motifs↗
PubMed2004

HCV genotype 4--an emerging threat as a cause of chronic liver disease in Indian (south) patients.

BACKGROUND: Hepatitis C virus (HCV) genotyping is relevant for the delivery of effective antiviral therapy. HCV genotypes are geographically restricted with genotype 4, which is resistant to therapy, traditionally considered to be confined to the Middle East and Africa. We report here on the occurrence of HCV genotype 4 in Indian (South) patients. OBJECTIVES: 1) To highlight the occurrence of HCV genotype 4 in the patient population attending a tertiary care hospital in south India. 2) To ascertain the difference in HCV viral loads and alanine aminotransferase (ALT) values between patients infected with HCV genotype 4 and those infected with the other two most commonly detected genotypes in this patient population viz., HCV genotypes 1 and 3. 3) To assess the genetic relatedness of the Indian strains to Genbank sequences, which we report for the first time. STUDY DESIGN: The study group consisted of 125 HCV infected, untreated patients who had been genotyped using type specific primers. Eight of the nine samples classified as HCV genotype 4 by this technique were subjected to nucleotide sequencing. Viral load estimations were carried out. Information on possible risk factors and ALT values were obtained from hospital records. Statistical analyses were carried out to compare viral loads and ALT values across genotypes. A phylogenetic tree was constructed and the genetic relatedness of the strains was assessed through sequence analysis. RESULTS: HCV genotype 4 was detected in nine of 125 (7.2%) patients. Eight of the nine were subjected to nucleotide sequencing and all strains were confirmed as HCV genotype 4. Six of the eight strains were closely related, with two strains being phylogenetically diverse. CONCLUSIONS: HCV genotype 4 is detected in a significant minority of HCV infected patients in India. This finding should be considered in designing strategies prior to initiation of therapy in Indian patients infected with HCV.

Adult↗