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At least 1,513 records · Page 84Linked to original sources
PubMed1998

Editing site recognition in plant mitochondria: the importance of 5'-flanking sequences.

Cytidine to uridine (C-to-U) editing occurs in plant mitochondria with very high specificity such that only specific cytidines are converted to uridines. The mechanisms for editing site selection in plant mitochondria are unknown. In order to examine the determinants of editing site recognition, repeated mitochondrial DNA sequences that include edited nucleotides have been evaluated as editing substrates. During evolution the maize mitochondrial ribosomal protein subunit 12 (rps12) gene recombined with intron 1 of the ribosomal protein subunit 3 (rps3) gene and a region of the S1-like sequence of the 2.3 kb plasmid. These recombinations created a second copy of an internal portion of the rps12 gene, known as rps12b, which includes the first four editing sites of rps12 transcripts. The duplicated sequence extends seven nucleotides upstream of editing site 1 and six nucleotides downstream from editing site 4. The sequences of rps12 and rps12b are identical between these sites except for a single change at -5 from editing site 1. These modifications did not effect C-to-U conversion at editing sites 2, 3, or 4 in rps12b; however, no editing was detected at editing site 1 in rps12b cDNAs. Thus, the 5' recombination abolished editing at site I, while the 3' recombination modified the downstream RNA sequence, but did not effect editing at site IV. Secondary structure prediction suggests that changes in editing site recognition do not correlate with differences in secondary structures, and that primary RNA sequence may be responsible for editing site specification.

Amino Acid Sequence↗
PubMed1991

Enteroviruses and idiopathic dilated cardiomyopathy.

Previous studies have suggested that some cases of idiopathic dilated cardiomyopathy (IDC) are due to persistent viral infection following an episode of viral myocarditis. Viral RNA sequences have recently been detected in material from patients with IDC using molecular biological techniques. We tested 40 samples from recipients' hearts explanted at cardiac transplantation for the presence of enteroviral RNA sequences, using a Northern blotting technique. Material from 19 cases of IDC and 21 cases of non-cardiomyopathic cardiac failure was examined together with Coxsackie-virus-infected neonatal mouse heart as a positive control and non-infected adult mouse heart as a negative control. A sharp band of viral RNA was detected in the positive control sample. No hybridization signal attributable to viral RNA was obtained for the negative control or for any of the test samples. We conclude that the role of enteroviruses in the pathogenesis of cardiomyopathy is not fully established and that further study is warranted.

Adult↗
PubMed1986

Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing.

Adenovirus Early Proteins↗
PubMed1995

Design of artificial short-chained RNA species that are replicated by Q beta replicase.

Different RNA species that are replicated by Q beta replicase have related secondary structures: for both plus and minus strands, "leader" stem structures were found at their 5' termini, while their 3' termini were unpaired. Parallel structures in complementary strands rather than antiparallel ones require the occurrence of wobble pairs and other imperfections in the stem regions. To test whether the leader structures are required for replication, artificial RNA sequences were synthesized by transcription from synthetic oligodeoxynucleotides with T7 RNA polymerase and assayed for their ability to be replicated by Q beta replicase. A synthetic short RNA species known to be replicated was amplified, forming a stable quasi-species; i.e., its sequence was conserved during hundreds of replication rounds. A synthetic mutant of this sequence that stabilized the leader in one strand but favored a 3'-terminal stem in the other one led to the complete loss of template activity. When new RNA sequences with the described structural requirements were designed and synthesized, their template activity was too low to be directly measurable; however, incubation with replicase produced replicating RNA whose sequence was closely related to the synthesized RNA species. The most likely interpretation is that the designed sequences were in a low montainous region in the replication fitness landscape and were optimized during amplification by Q beta replicase to a nearby fitness peak. The structural features postulated to be required for replication were not only conserved but even improved in the outgrowing mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2003

Differences in Ibaraki virus RNA segment 3 sequences from three epidemics.

Phylogenetic tree and partial nucleotide sequence analysis of RNA segment 3 were conducted to compare the Ibaraki virus (IBAV) strains from three epidemics in Japan, and serotype 2 epizootic hemorrhagic disease virus strains isolated in Australia, Taiwan, and Canada. Each strain was classified relative to the Ibaraki disease (IBAD) epidemics, which occurred in 1959-1960, 1987, or 1997-1998. In particular, major variation of the gene was identified in the strains isolated after 1997 when a new type of IBAD with the abnormal birth was confirmed. Ibaraki viruses isolated in Japan were more closely related to Taiwanese and Australian strains based on genetics, while the Canadian strain was more distantly related.

Animals↗
PubMed1979

Characterization and transcription analysis of a cloned sequence derived from a major developmentally regulated mRNA of D. discoideum.

The plasmid pDd 812 contains a portion of a poly(A)+ RNA sequence isolated from developing cells of the cellular slime mold Dictyostelium discoideum (Williams and Lloyd, 1979). The poly(A)+ RNA complementary to this plasmid shows an increase in concentration during the first 4 hr of development followed by a decrease in concentration during the following 4 hr. This RNA is very abundant after 3-4 hr of development, constituting at least 2% of the poly(A)+ RNA population. In this study, we demonstrate that this poly(A)+ RNA is an mRNA sequence by translating the RNA complementary to pDd 812 in a heterologous system. The mRNA directs the synthesis of a major polypeptide of 33,000 daltons and a minor polypeptide of 31,000 daltons. We have used the plasmid DNA immobilized on filters to analyze the transcription of this RNA sequence in isolated nuclei. The amount of transcript synthesized in nuclei isolated at various stages of development which was complementary to pDd 812 changed in the same way as did the cytoplasmic concentration of this RNA--that is, maximal transcription occurred after 3-4 hr of development. Because this result was observed using labeling periods as short as 5 min, we believe that this change is unlikely to reflect a change in the rate of processing of RNA. We interpret these results to indicate that, at least in part, the control of the synthesis of this RNA is at the level of gene transcription.

Base Sequence↗
PubMed2026

REG3α is a Predictive Biomarker of Complicated Disease from Preclinical through Established Crohn's Disease.

BACKGROUND: Regenerating islet-derived 3-alpha (REG3&#x3b1;) is a serum biomarker in patients with graft-versus-host disease (GVHD) linked to 6-month mortality. REG3&#x3b1; is produced by intestinal Paneth cells, which are implicated in Crohn's disease (CD) pathophysiology. OBJECTIVE: To assess associations between serum REG3&#x3b1; and progressive CD DESIGN: Serum REG3&#x3b1; was measured in two cross-sectional (M: Mount Sinai, L: Leuven) and a pre-diagnostic cohort (P: PREDICTS) with serial samples up to 10 years before CD diagnosis. Tissue REG3&#x3b1; expression was assessed via bulk RNA sequencing from paired ileal and colonic biopsies. Serum REG3&#x3b1; and tissue REG3&#x3b1; were associated with CD progression (hospitalization, surgery, steroid course, or new advanced therapy). Single-cell RNA sequencing data explored associations between REG3&#x3b1; expression, Paneth cell phenotypes, and CD. RESULTS: In 394 patients, high serum REG3&#x3b1; associated with CD progression, independent of C-reactive protein and endoscopic activity (M: HR 1.9 (95%CI 1.3-2.8); L: HR 2.9 (95%CI 1.9-4.6), both p<0.001). The association persisted in patients with mild or inactive CD. In the pre-diagnostic cohort, high serum REG3&#x3b1; predicted the development of CD, particularly complicated (B2/3) and surgical presentations, up to 10 years before diagnosis (P). Analysis of REG3&#x3b1; expression and Paneth cell transcriptomes suggested that CD is associated with loss of regenerative Paneth cell populations and enrichment in REG3&#x3b1;-expressing populations, suggesting a mechanism through which changes in serum REG3&#x3b1; associate with complicated CD. CONCLUSION: Serum REG3&#x3b1; holds potential as a non-invasive, prognostic biomarker in CD, independent of disease activity. High serum REG3&#x3b1;, even years before diagnosis, is linked to a complicated disease course.

Crohn&#x2019;s Disease↗
PubMed1997

Affinity purification of RNA: sequence-specific capture by nonionic morpholino probes.

Nucleic acid isolation for amplification-based diagnostics requires techniques that do not co-purify inhibitors of DNA polymerases. Also, other requirements for an ideal sample preparation technology include ease of use, capability for automation, high recovery and the use of nontoxic reagents. Affinity purification techniques provide high purification factor with minimal sample processing. Hybridization is the affinity interaction specific to nucleic acids and thus provides a uniquely advantageous method for purifying DNA or RNA for subsequent manipulation. Nonionic (morpholino) probes (Neu-Probes, AntiVirals, Corvallis, OR, USA) have several unique hybridization properties, including resistance to nucleases and the ability to hybridize independently of salt concentration. Therefore, such probes provide advantages over DNA probes for sample preparation by hybridization capture. Three formats for hybridization-based purification of human immunodeficiency virus (HIV) RNA were evaluated using RNA transcripts spiked into crude lysates of normal human plasma. Indirect capture used streptavidin-coated microparticles to capture hybrids of biotinylated capture probes and HIV RNA. Direct capture used particles precoated with probes. In addition, a novel method for acceleration of sequence-specific hybridization was developed and shown to give consistently high recoveries.

DNA-Directed DNA Polymerase↗
PubMed1980

Polysomal poly(A)+ RNA in liver of rats fed 3'-methyl-4-dimethylaminoazobenzene and in hepatoma induced by the same carcinogen.

Feeding of carcinogenic azo dyes to rats results in a release into the cytoplasm of RNA sequences which in liver cells of control animals are degraded in the cell nucleus. A cross-hybridization of polysomal poly(A)+ RNA from liver of rats fed the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene and from liver of control animals with their complementary DNA has shown, that the disruption of the processing and/or release of nuclear RNA induced by the carcinogen is not reflected in a change in the polysomal poly(A)+ RNA. After 17 weeks of feeding the hepatocarcinogen, there is no difference in the sequence complexity of polysomal poly(A)+ RNA in the liver. It is therefore not probable that the RNA sequences released from the nucleus by the azo dye serve as a template for protein synthesis. An alteration in the polysomal poly(A)+ RNA population was observed only temporarily at an earlier stage of feeding of the azocarcinogen. It coincided with the regeneration of the liver in a response to the initial toxic effect of the azocarcinogen. Therefore, it is probable that this alteration is the result of a temporary change in the population of liver cells. A cross-hybridization of liver and hepatoma complementary DNA with the polysomal poly(A)+ RNA from both organs have shown an overlap of the polysomal poly(A)+ RNA sequences of the hepatoma with the sequences present in the liver, with many liver sequences missing, or present only in very low concentration in the hepatoma.

Animals↗
PubMed2003

Sequences downstream of the 5' splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA.

Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5' splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a "kissing-loop" mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5' splice donor site, are required for efficient RNA packaging and dimerization.

Animals↗
PubMed2001

Poly(A) polymerase and the regulation of cytoplasmic polyadenylation.

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.

Animals↗
PubMed1996

Multiple requirements for nematode spliced leader RNP function in trans-splicing.

The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.

Animals↗
PubMed1997

Characterization of fluoranthene- and pyrene-degrading Mycobacterium-like strains by RAPD and SSU sequencing.

Bacterial isolates were obtained from two sites in New Zealand contaminated with polycyclic aromatic hydrocarbons. Isolates capable of degrading polycyclic aromatic hydrocarbons were characterized in two mycobacterial groups according to phenotypic properties. These groups were supported by random amplified polymorphic DNA analysis. Nucleotide sequences of 16S ribosomal RNA genes from isolates representing each group were determined and compared with other mycobacterial 16S ribosomal RNA sequences. The taxonomic relationships of these isolates are considered.

Biodegradation, Environmental↗
PubMed1995

In vitro selection of RNA aptamers that can bind specifically to Tat protein of HIV-1.

The Tat protein of human immunodeficiency virus interacts specifically with its target RNA sequence, TAR, and activates viral gene expression at the early stage of infection. Here, we have used in vitro genetic selection strategy to determine what sequence or structural motifs might exist between RNA's that interact specifically with the Tat protein. Starting from a RNA pool that has a 120 base random sequence core, aptamers that bind specifically to the Tat protein were selected by repeating 11 rounds of selection and amplification. A comparative analysis of 64 aptamers that were isolated from the 11th generation revealed two main sequence classes. Interestingly, one of these two classes of aptamers had minimum of one U residues in bulge loop and 2 specific adjacent base pairs. This region is very much homologous to the core sequence of TAR RNA that is essential for the specific Tat-TAR interactions. Further analyses of the sequences from the 11th generation should reveal what kind of RNA structures are required in order to show a high affinity for the Tat protein.

Base Sequence↗
PubMed2026

Pan-cancer characterization of HMGA1 reveals its oncogenic role in tumor microenvironment and stemness: functional validation in pancreatic cancer migration and invasion.

BACKGROUND: HMGA1 is a chromatin-associated oncogenic factor implicated in tumor progression, epithelial-mesenchymal transition (EMT), stemness, and metastasis. However, its pan-cancer expression and prognostic patterns, epigenetic activation, and relationship with malignant-cell stemness/plasticity and tumor microenvironment (TME) remodeling in pancreatic adenocarcinoma (PAAD) remain incompletely defined. This study aimed to systematically characterize HMGA1 across cancers and clarify its clinical and biological relevance in PAAD. METHODS: Pan-cancer transcriptomic, clinical, genetic, methylation, immune, and stemness data were integrated from multiple public databases. PAAD single-cell RNA sequencing data were analyzed to localize HMGA1 expression, infer malignant-cell pseudotime, calculate a stemness module score, and assess ligand-receptor communication using CellChat. Public HMGA1-knockdown RNA sequencing data were reanalyzed to evaluate transcriptional remodeling. The Cancer Genome Atlas (TCGA)-PAAD expression and methylation data were used to assess TME-remodeling, immune-suppression, stemness/plasticity, cytokine/chemokine, checkpoint, and promoter-methylation features. HMGA1 expression and function were further examined using immunohistochemistry (IHC), quantitative real-time polymerase chain reaction, Western blotting, wound-healing assays, and Transwell migration and invasion assays. RESULTS: HMGA1 was upregulated in most tumor types, and high expression was associated with unfavorable survival in multiple cancers, including PAAD. In PAAD, HMGA1 was enriched in malignant epithelial cells and positively correlated with pseudotime (Spearman's rho =0.594), while the stemness module score increased along pseudotime (rho =0.748). HMGA1-high malignant cells showed markedly stronger CellChat-inferred outgoing communication, predominantly involving extracellular matrix (ECM)-receptor, adhesion-related, and selected immunomodulatory ligand-receptor axes. HMGA1 knockdown was associated with broad remodeling of EMT, TGF-&#x3b2;, Hedgehog, IL6/JAK/STAT3, KRAS, and cancer stem cell/stemness-related programs rather than uniform suppression of these programs. HMGA1 promoter methylation was inversely correlated with HMGA1 expression (rho =-0.633) and the TME-remodeling score (rho =-0.347). HMGA1 was associated with selected mediators, including PPIA, PLAU, ANXA1, LGALS9, TGFB1, CD276, and CD47, but not with a generalized checkpoint-high phenotype. Functionally, HMGA1 knockdown significantly reduced pancreatic cancer (PC) cell migration and invasion. CONCLUSIONS: These findings support an association-based model in which promoter hypomethylation-associated HMGA1 activation is linked to malignant epithelial stemness/plasticity, ECM/adhesion-dominant TME remodeling, selected immunomodulatory programs, and aggressive PAAD phenotypes. Further mechanistic and clinical validation is required before HMGA1 can be used for therapeutic stratification or immunotherapy-response prediction.

HMGA1↗
PubMed2003

Gemella bacteraemia characterised by 16S ribosomal RNA gene sequencing.

AIMS: To define epidemiology, clinical disease, and outcome of gemella bacteraemia by 16S rRNA gene sequencing. To examine the usefulness of the Vitek, API, and ATB systems in identifying two gemella species. METHODS: All alpha haemolytic streptococci other than Streptococcus pneumoniae isolated from blood cultures during a six year period were identified by conventional biochemical methods, the Vitek system, and the API system. 16S rRNA gene sequencing was performed on all isolates identified by both kits as gemella with >or= 95% confidence or by either kit as any bacterial species with < 95% confidence. The ATB expression system was used to identify the two isolates that were defined as gemella species by 16S rRNA gene sequencing. RESULTS: Of the 302 alpha haemolytic streptococci other than S pneumoniae isolated, one was identified as Gemella morbillorum, and another as Gemella haemolysans by 16S rRNA gene sequencing. The patient with monomicrobial G morbillorum bacteraemia was a 66 year old man with community acquired infective endocarditis with septic thromboemboli. The patient with G haemolysans bacteraemia was a 41 year old woman with hospital acquired polymicrobial bacteraemia during the neutropenic period of an autologous bone marrow transplant for non-Hodgkin's lymphoma, the first case of its kind in the English literature. The API and ATB expression systems only identified the second strain as G haemolysans at 94% and 99% confidence, respectively, whereas the Vitek system identified none of the two strains correctly at > 70% confidence. CONCLUSIONS: Gemella bacteraemia is uncommon. 16S rRNA gene sequencing is the method of choice for identification of gemella and gemella-like isolates.

Adult↗
PubMed1990

Molecular analysis of a U3 RNA gene locus in tomato: transcription signals, the coding region, expression in transgenic tobacco plants and tandemly repeated pseudogenes.

By screening a tomato genomic library with a tomato U3 RNA probe, we detected a U3 genomic locus whose coding region was determined by primer extension (5' end) and direct RNA sequencing of purified U3 RNA from tomato (3' end). Tomato U3 RNA is 216 nucleotides long, contains all the four evolutionarily highly conserved sequence blocks (Boxes A to D), has at its 5' end a cap not precipitable with anti-m3G antibodies and can be folded into a peculiar secondary structure with two stem-loops at its 5' end. A tagged derivative of the U3 gene was faithfully expressed in transgenic tobacco plants. In the 5' flanking region both plant-specific UsnRNA transcription signals [the TATA-like sequence and the upstream sequence element (USE)] were present, but were positioned closer to each other and also to the cap site in the U3 gene than in the genes for the plant spliceosomal UsnRNAs studied so far. The 3' flanking region of the tomato U3 gene lacked the consensus sequence of the putative termination signal established for the plant spliceosomal UsnRNA genes and contained a pyrimidine-rich tract (R1) followed by four tandemly repeated U3 pseudogenes (U3.1 ps to U3.4 ps) flanked by slightly altered forms (R2 to R5) of R1 and most probably generated by DNA-mediated events. Our results are in line with the conjecture that the enzyme transcribing the tomato U3 gene has different structural requirements for transcriptional activity than the enzyme transcribing plant U1, U2 and U5 genes.

Base Sequence↗