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At least 1,603 records · Page 89Linked to original sources
PubMed2004

Sigma54 enhancer binding proteins and Myxococcus xanthus fruiting body development.

A search of the M1genome sequence, which includes 97% of the Myxococcus xanthus genes, identified 53 sequence homologs of sigma54-dependent enhancer binding proteins (EBPs). A DNA microarray was constructed from the M1genome that includes those homologs and 318 other M. xanthus genes for comparison. To screen the developmental program with this array, an RNA extract from growing cells was compared with one prepared from developing cells at 12 h. Previous reporter studies had shown that M. xanthus has initiated development and has begun to express many developmentally regulated genes by 12 h. The comparison revealed substantial increases in the expression levels of 11 transcription factors that may respond to environmental stimuli. Six of the 53 EBP homologs were expressed at significantly higher levels at 12 h of development than during growth. Three were previously unknown genes, and they were inactivated to look for effects on fruiting body development. One knockout mutant produced fruiting bodies of abnormal shape that depended on the composition of the medium.

Amino Acid Sequence↗
PubMed2003

Molecular assays for detection of human metapneumovirus.

The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

DNA Primers↗
PubMed2000

Natural genetic exchanges between vaccine and wild poliovirus strains in humans.

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3' moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.

Adolescent↗
PubMed2002

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs.

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Flowers↗
PubMedDate not supplied

[RNA isolation and purification methods].

Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.

Blotting, Northern↗
PubMed1992

Extrachromosomal ribosomal DNA of Didymium iridis: sequence analysis of the large subunit ribosomal RNA gene and sub-telomeric region.

The ribosomal DNA of the myxomycete Didymium iridis is organized as extrachromosomal linear molecules of about 20 kb, containing only one transcription unit of the ribosomal RNA genes. We have determined the sequence of the large subunit ribosomal RNA (LSU rRNA) gene as well as the sub-telomeric and telomeric regions. The LSU rRNA gene was found to encode a 3857 nucleotide-long LSU rRNA, interrupted by a transcribed spacer and two group I introns. A complete secondary structure model of D. iridis LSU rRNA has been constructed. The compact sub-telomeric region of D. iridis rDNA was found to contain several directly repeated sequence elements that include the simple telomere motif TTAGGG. Based on pairwise comparisons of LSU rRNA sequences, the time of divergence between the two myxomycete genera Didymium and Physarum was estimated.

Animals↗
PubMed2006

Perkinsoide chabelardi n. gen., a protozoan parasite with an intermediate evolutionary position: possible cause of the decrease of sardine fisheries?

Phenotypic scrutiny on the life cycle of Icthyodinium chabelardi (Perkinsoide chabelardi n. gen.) based on ultrastructural techniques, and molecular phylogenetic analysis of RNA gene sequences, were carried out in order to elucidate the taxonomic position of this parasite. The absence of plastid, presence of trichocysts, and chromosomes or chromatin condensed and low in number, suggested that this protozoan could be considered a dinoflagellate syndinial parasite. However, the life cycle, schizogonic divisions and structure of schizonts inside the host, the nuclei without the typical dinoflagellate appearance, presence of rhoptrias-like structures, a possible pseudo-conoid, and the biflagellated spore, resembled those of the genus Perkinsus. Phylogenetic analysis of genes transcribing for the RNA forming the small subunit and the large subunit suggests that this parasite has an ambiguous evolutionary position within the group formed by dinoflagellates, perkinsids and syndinials. Because of differences with dinoflagellates and similarities with perkinsids, we propose to change the generic name to P. chabelardi n. gen. High stationary infection prevalence on Sardina pilchardus eggs was observed. This protozoan parasite caused the death of all the infected sardine eggs, and therefore a high impact in the recruitment of this fishery in the Atlantic coast is expected.

Animals↗
PubMed2025

Spatial transcriptomics-aided localization for single-cell transcriptomics with STALocator.

Single-cell RNA-sequencing (scRNA-seq) techniques can measure gene expression at single-cell resolution but lack spatial information. Spatial transcriptomics (ST) techniques simultaneously provide gene expression data and spatial information. However, the data quality of the spatial resolution or gene coverage is still much lower than the quality of the single-cell transcriptomics data. To this end, we develop a ST-Aided Locator for single-cell transcriptomics (STALocator) to localize single cells to corresponding ST data. Applications on simulated data showed that STALocator performed better than other localization methods. When applied to the human brain and squamous cell carcinoma data, STALocator could robustly reconstruct the relative spatial organization of critical cell populations. Moreover, STALocator could enhance gene expression patterns for Slide-seqV2 data and predict genome-wide gene expression data for fluorescence in situ hybridization (FISH) and Xenium data, leading to the identification of more spatially variable genes and more biologically relevant Gene Ontology (GO) terms compared with the raw data. A record of this paper's transparent peer review process is included in the supplemental information.

Single-Cell Analysis↗
PubMed2026

Spatial Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Tissue by spRandom-seq.

The molecular pathogenesis of infectious diseases and cancer is orchestrated by nanoscale of host and microbial RNA transcripts within the tissue microenvironment. Nevertheless, spatially resolving the comprehensive transcriptional landscape within complex clinical tissues, like formalin-fixed paraffin-embedded (FFPE) specimens, still poses a formidable challenge. Here, we present spRandom-seq, a random primer-based spatial total RNA sequencing technology designed to spatially resolve complete transcriptomes from host, bacteria, and even nanoscale viruses in FFPE tissues. Capitalizing on the random primer design, our technology not only facilitated the discovery of specific lncRNAs and alternative splicing events in mouse brain and olfactory bulb, but also delineated pronounced spatial heterogeneity in clinical FFPE sections-across distinct tumor regions in breast cancer and microbial infection sites in Klebsiella pneumoniae-infected tissues. Importantly, integrated analysis of host and viral RNAs in FFPE samples from hepatitis B virus (HBV)‑positive hepatocellular carcinoma (HCC) demonstrated that complement and coagulation pathways were specifically activated across expansive HBV‑infected tumor areas, which also exhibited an increased burden of copy number variations (CNVs). Owing to its compatibility with existing spatial transcriptomics platforms and minimal operational complexity, spRandom-seq represents a practical and scalable approach for clinical pathology applications and infection diagnostics.

Paraffin Embedding↗
PubMed1993

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.

Base Sequence↗
PubMed2003

Analysis of ribosomal RNA intergenic spacer (IGS) sequences in species and populations of Gyrodactylus (Platyhelminthes: Monogenea) from salmonid fish in Northern Europe.

The intergenic spacer (IGS) region of ribosomal RNA genes was amplified and sequenced from a variety of Gyrodactylus specimens collected from wild and farmed Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, and grayling Thymallus thymallus, from various locations in Northern Europe. Phylogenetic analysis of the sequences confirmed the distinction between G. salaris Malmberg, 1957 and G. thymalli Zitnan, 1960, supporting their validity as separate species. G. salaris adapted to rainbow trout are also distinct from the parasites found on Atlantic salmon, supporting the existence of a rainbow-trout form that was initially identified on the basis of morphological differences. Analysis of the IGS did not provide good resolution of different populations of G. salaris sensu stricto, but was consistent with epidemiological evidence which indicates that introduction of the parasite to Norway was recent and limited. The IGS may be helpful in distinguishing forms of G. salaris that are pathogenic to Atlantic salmon from those that are not.

Animals↗
PubMed1984

Cloning and sequence analysis of the Escherichia coli 4.5 S RNA gene.

The structure of the Escherichia coli gene coding for the metabolically stable 4.5 S RNA has been determined by cloning and DNA sequence analysis. Results from Southern hybridization assays carried out prior to cloning show the 4.5 S DNA to be limited to a single locus in the E. coli K12 genome. A 5.4 X 10(3) base DNA fragment containing the 4.5 S DNA was cloned into plasmid pBR322 for restriction, hybridization and sequence analyses. Cells harboring the cloned gene overproduce the 4.5 S RNA by 15-fold under normal culturing conditions; however, no effect on growth rate is observed. DNA sequencing revealed only one copy of the 4.5 S RNA gene, with a deduced RNA sequence both longer at 114 bases and slightly different from the RNA sequence reported earlier. A promoter structure immediately preceding the structural gene shows good agreement with the prokaryotic consensus sequence at both the -35 and -10 regions. In addition, a G + C-rich sequence between the Pribnow box and the start of transcription agrees well with an apparent consensus sequence found for other stable RNA genes also under stringent control. No clearly recognizable termination signal was found immediately downstream from the 3' terminus of the 4.5 S DNA, although structural elements with that potential appear to occur. A potential coding sequence for a protein occurs about 100 bases downstream from the 4.5 S DNA, suggesting the possibility of a dual function 4.5 S RNA-mRNA transcript.

Base Sequence↗
PubMed1995

Nucleotide sequence and characterization of the large mitochondrial rRNA gene of Penicillium urticae, and its comparison with those of other filamentous fungi.

The nucleotide sequence of a large rRNA gene and its flanking regions in cloned fragments of mitochondrial DNA from a patulin producer, Penicillium urticae NRRL2159A, was determined by dideoxy sequencing, and the 5' end and intron-exon border of the 1-rRNA gene were determined by primer extension analysis and RNA sequencing, respectively. In addition to the extensive sequence homology of the 3' end of the P. urticae mt 1-rRNA gene with those of Aspergillus nidulans and Neurospora crassa, the P. urticae gene had a 1,685 bp intron which separates a 3,307 bp 5' exon and a 583 bp 3' exon. In spite of being closely related Penicillium species, the size of the 5' exon of the P. urticae mt 1-rRNA is 472 bp larger than that of P. chrysogenum, whereas the sizes of the 3' exon and intron of P. urticae are very similar to those of P. chrysogenum (581 bp for the 3' exon and 1,678 bp for the intron). The intron of P. urticae contains a structure similar to the consensus one of the self splicing group IA intron and a large open reading frame suggested to be a gene for ribosomal protein S5. A sequence similar to the I-SceI recognition sequence was found at the exon-intron border. Extensive sequence homology was observed between P. urticae and P. chrysogenum, exceptions being in four regions in the 5' exon. These non-homologous regions were located in the hairpin and variable regions outside of the core structures. Comparison of the mt 1-rRNA sequences of several filamentous fungi revealed that the above four non-homologous regions are greatly expanded, and two other non-homologous regions appear at the 3' ends of the 5' exon and 3' exon.

Aspergillus nidulans↗
PubMed1996

Phylogenetic analysis of the envelope gene of Japanese encephalitis virus.

Variation among Japanese encephalitis virus (JEV) strains has been documented in a number of studies by employing a variety of techniques like HI, NT, CF, RNA fingerprinting and sequencing of prM region. We report the complete envelope (E) gene sequence and the deduced amino acid sequence of four strains of JEV from the Indian subcontinent. These sequences were compared with published E gene sequences of 16 strains of JEV. Pairwise comparisons of the E gene nucleotide and deduced amino acid sequences of these strains indicated an overall sequence conservation. A majority of the differences in the four strains were located in domain A and domain C (Mandl et al., 1989). Phylogenetic analysis of the E gene sequences by a variety of tree building methods identified four clusters. Viral groupings did not correspond to geographic origin, isolation host or virulence. Evidence for positive selection operating on some strains belonging to different clusters was obtained.

Animals↗
PubMed1999

[A novel alternative splicing mode of the erythropoietin receptor gene in rats].

OBJECTIVE: To detect new alternative splicing mode of erythropoietin receptor (EpoR) gene in normal rats. METHODS: Total RNAs were isolated from bone marrow mononuclear cells of normal Wistar rats. Reverse transcriptase polymerase chain reaction (RT-PCR) of the total RNAs was done and the amplified products were sequenced. RESULTS: After RT-PCR with a pair of primers (sense: 637/657 bp, and antisence: 903/882 bp), two bands were detected, the expected band (267 bp) and a longer one (346 bp) which had a 79 bp insert between 736th and 737th bases from the first ATG of the rat EpoR nucleotide. Sequence analysis showed that the 79 bp insert was the retaining intron 5 with a C-to-T point mutation at 16th base and an insertion of G base between 49th and 50th base. The insertion of G base caused a shift in reading frame and resulted EpoR with a truncated intracellular domain. CONCLUSION: A new alternative splicing of EpoR mRNA encoding truncated intracellular domain was identified in normal Wistar rats. Its implication in rat erythropoiesis remains to be determined.

Alternative Splicing↗
PubMed2002

Global gene expression profiling of end-stage dilated cardiomyopathy using a human cardiovascular-based cDNA microarray.

To obtain a genomic portrait of heart failure derived from end-stage dilated cardiomyopathy (DCM), we explored expression analysis using the CardioChip, a nonredundant 10,848-element human cardiovascular-based expressed sequence tag glass slide cDNA microarray constructed in-house. RNA was extracted from the left ventricular free wall of seven patients undergoing transplantation, and five nonfailing heart samples. Cy3- and Cy5-labeled (and reverse dye-labeled) cDNA probes were synthesized from individual diseased or nonfailing adult heart RNA, and hybridized to the array. More than 100 transcripts were consistently differentially expressed in DCM >1.5-fold (versus pooled nonfailing heart, P < 0.05). Atrial natriuretic peptide was found to be up-regulated in DCM (19-fold compared to nonfailing, P < 0.05), as well as numerous sarcomeric and cytoskeletal proteins (eg, cardiac troponin, tropomyosin), stress response proteins (eg, HSP 40, HSP 70), and transcription/translation regulators (eg, CCAAT box binding factor, eIF-1AY). Down-regulation was most prominently observed with cell-signaling channels and mediators, particularly those involved in Ca(2+) pathways (Ca(2+)/calmodulin-dependent kinase, inositol 1,4,5-trisphosphate receptor, SERCA). Most intriguing was the co-expression of several novel, cardiac-enriched expressed sequence tags. Quantitative real-time reverse transcriptase-polymerase chain reaction of a selection of these clones verified expression. Our study provides a preliminary molecular profile of DCM using the largest human heart-specific cDNA microarray to date.

Adult↗
PubMed1989

Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae.

The complete nucleotide sequence of the large (L) genome segment of Bunyamwera virus has been determined from overlapping cDNA clones. The segment is 6875 nucleotides long and has a base composition of 29.8% A, 17.9% C, 15.4% G, and 36.9% U. Eighteen of the terminal 19 nucleotides at the 3' and 5' ends are complementary. In the viral-complementary (+ sense) RNA there is a single long open reading frame (ORF) from AUG at bases 51-53 to a UAG stop codon at bases 6765-6767; this ORF encodes a polypeptide of 2238 amino acids (MW 259,000), corresponding to the L protein which has been mapped to the L RNA segment by analysis of reassortants of Bunyamwera, Batai, and Maguari viruses. The amino-terminal 46 amino acids of the L protein show strong homology (63% identity) with the amino-termini of ORFs predicted from limited sequence analysis of the L segments of La Crosse and snowshoe hare bunyaviruses. Comparison with the polymerase proteins encoded by other negative-strand viruses showed weak homology with part of the influenza virus PB1 protein, but no homology was detected with the other influenza virus polymerase proteins nor with the L proteins of arenaviruses, paramyxoviruses, and rhabdoviruses. At the 5' end of genomic (- sense) RNA there is an AUG-initiated ORF potentially encoding a protein of 14,700; the significance of this ORF is unknown at present.

Amino Acid Sequence↗
PubMed2006

Analysis of Affymetrix GeneChip data using amplified RNA.

When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5' end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic. Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting.

Algorithms↗