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Results for “RNA sequencing analysis”

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At least 541 records · Page 30Linked to original sources
PubMed1998

Self-cleaving circular RNA associated with rice yellow mottle virus is the smallest viroid-like RNA.

We report the sequence, structural features, and self-cleaving activity of the small circular RNA (sc-RNA) associated with rice yellow mottle sobemovirus (RYMV). At 220 nucleotides, the RYMV sc-RNA represents the smallest naturally occurring viroid-like RNA currently documented in the literature. It is similar to other circular satellite RNAs (sat-RNAs) and viroids in being G-C-rich with a high level of self-complementarity. The predicted native structure is essentially a rod with one branched terminus. A region of the RYMV sc-RNA, constituting 24% of the sequence, exhibits 89% identity to the sat-RNA associated with the Australasian isolates of lucerne transient streak sobemovirus. This region is also structurally similar in all three RNAs in that it forms the left terminus of each rod. Dimeric runoff transcripts of cloned RYMV sc-RNA undergo efficient autocatalytic in vitro cleavage in the (+) but not the (-) polarity. Analysis of the (+) sequence indicates the presence of a hammerhead ribozyme resembling that of carnation small retroviroid-like RNA and the genomic satellite transcript of newt. Inefficient cleavage of (+) monomeric transcripts, and a short stem III in the hammerhead, are features consistent with a double-hammerhead mode of self-cleavage. The presence of sat-RNA and retroviroid-like structures within a single RNA suggests a possible role for the RYMV sc-RNA as an evolutionary intermediate between these subviral RNAs.

Base Sequence↗
PubMed1973

Structural characteristics and nucleotide sequence analysis of genomic RNA from RD-114 virus and feline RNA tumor viruses.

The results of molecular hybridization experiments have demonstrated that the RNA genome of RD-114 virus has extensive nucleotide sequence homology with the RNA genome of Crandell virus, an endogenous type C virus of cats, but only limited homology with the RNA genomes of feline sarcoma virus and feline leukemia virus. The genomic RNAs of RD-114 virus and Crandell virus also had identical sedimentation coefficients of 50S. A structural rearrangement of genomic RNA did not exist within released RD-114 virions, whereas a structural rearrangement of genomic RNA did occur within feline sarcoma virions and feline leukemia virions after release from virus-producing cells.

Animals↗
PubMed1994

Nucleotide sequence statistical analysis of pauses in RNA elongation by Escherichia coli RNA polymerase.

A convenient motif-searching program has been developed, based on a double correlation algorithm, for analysis of the pulse character of Escherichia coli transcription. Activity in the zone of minimal pause formation (-1,2 bp) is precisely determined. Oligonucleotides (di-, tri- and tetranucleotides) are randomized by their pause-generating activity. 'CATG' and 'CATGC' are detected which coincide with the primary structure of RNA associated with distinctive delays in biologically meaningful situations.

Algorithms↗
PubMed2006

GUUGle: a utility for fast exact matching under RNA complementary rules including G-U base pairing.

MOTIVATION: RNA secondary structure analysis often requires searching for potential helices in large sequence data. RESULTS: We present a utility program GUUGle that efficiently locates potential helical regions under RNA base pairing rules, which include Watson-Crick as well as G-U pairs. It accepts a positive and a negative set of sequences, and determines all exact matches under RNA rules between positive and negative sequences that exceed a specified length. The GUUGle algorithm can also be adapted to use a precomputed suffix array of the positive sequence set. We show how this program can be effectively used as a filter preceding a more computationally expensive task such as miRNA target prediction. AVAILABILITY: GUUGle is available via the Bielefeld Bioinformatics Server at http://bibiserv.techfak.uni-bielefeld.de/guugle

Algorithms↗
PubMed1999

In vitro selection of RNAs with increased tertiary structure stability.

An in vitro selection system was devised to select RNAs based on their tertiary structural stability, independent of RNA activity. Selection studies were conducted on the P4-P6 domain from the Tetrahymena thermophila group I intron, an autonomous self-folding unit that contains several important tertiary folding motifs including the tetraloop receptor and the A-rich bulge. Partially randomized P4-P6 molecules were selected based on their ability to fold into compact structures using native gel electrophoresis in the presence of decreasing concentrations of MgCl2. After 10 rounds of the selection process, a number of sequence alterations were identified that stabilized the P4-P6 RNA. One of these, a single base deletion of C209 within the P4 helix, significantly stabilized the P4-P6 molecule and would not have been identified by an activity-based selection because of its essential role for ribozyme function. Additionally, the sequence analysis provided evidence that stabilization of secondary structure may contribute to overall tertiary stability for RNAs. This system for probing RNA structure irrespective of RNA activity allows analysis of RNA structure/function relationships by identifying nucleotides or motifs important for folding and then comparing them with RNA sequences required for function.

Base Sequence↗
PubMedDate not supplied

Ascidian mitochondrial tRNA(Met) possessing unique structural characteristics.

Methionine tRNA was purified from muscle mitochondria of the ascidian Halocynthia roretzi and its RNA sequence was determined. Analysis of the nucleotide sequence revealed that unlike most metazoan mitochondrial tRNAs(Met), which have a highly conserved cytidine (C) or C-derivative at the wobble position, the H. roretzi mitochondrial tRNA(Met) possesses 5-carboxymethylaminomethyluridine (cmnm5U) at the first position of the anticodon. This is the first report of a single mitochondrial tRNA(Met) species having uridine (U) or a U-derivative at the wobble position.

Animals↗
PubMed1977

The use of nuclease P1 in sequence analysis of end group labeled RNA.

A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.

Base Sequence↗
PubMed1998

More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns.

Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.

Adenosine↗
PubMed2004

Simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by polymerase chain reaction-single strand conformation polymorphism and sequence analysis of the RNA polymerase gene (rpoB).

Interspecies variations and mutations associated with rifampin resistance in rpoB of Mycobacterium allow for the simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by PCR-SSCP analysis and PCR- sequencing. One hundred and ten strains of rifampin-susceptible M. tuberculosis, 14 strains of rifampin-resistant M. tuberculosis, and four strains of the M. avium complex were easily identified by PCR-SSCP. Of another seven strains, which showed unique SSCP patterns, three were identified as rifampin-resistant M. tuberculosis and four as M. terrae complex by subsequent sequence analysis of their rpoB DNAs (306 bp). These results were concordant with those obtained by susceptibility testing, biochemical identification, and 16S rDNA sequencing.

Antitubercular Agents↗
PubMed1990

Use of Chenopodium hybridum facilitates isolation of brome mosaic virus RNA recombinants.

Three mutant brome mosaic virus (BMV) RNA-2 transcripts bearing two alterations in the pseudoknot region and one in arm C of the 3' tRNA region, previously characterized as being deficient in tRNA-like functions, have been assayed for their ability to infect and replicate (in the presence of wild-type RNAs-1 and -3) in Chenopodium hybridum plants. Although the introduced mutations have been shown to incapacitate the replication of RNA-2 in barley protoplasts, C. hybridum plants inoculated with these mutants developed local lesions indistinguishable in appearance and morphology from control inoculations containing wild-type RNA-2. Sequence analysis of progeny RNA-2 from two single lesion isolates for each mutant inoculum revealed that the input mutations were restored to functional sequences by homologous recombination within the 3'tRNA-like region. These results, which reflect the ease with which progeny RNA can be characterized from single lesions, exemplify the value of C. hybridum for studying recombination among viral RNAs.

Base Sequence↗
PubMed1985

Cloning and nucleotide sequence analysis of transfer RNA genes from Mycoplasma mycoides.

As part of an investigation of the tRNA genes of Mycoplasma mycoides, two HindIII fragments of mycoplasma DNA comprising 0.4 and 2.5 kilobases (kb), respectively, were cloned in pBR322 and their nucleotide sequences determined. Only one tRNA gene was found in the 0.4 kb fragment, the gene for tRNAArg with the anticodon TCT, while the 2.5 kb fragment contained nine different tRNA genes arranged in a cluster which presumably constitutes a transcriptional unit. The clustered tRNA genes, with their respective anticodons, were as follows: Arg (ACG), Pro (TGG), Ala (TGC), Met (CAT), Ile (CAT), Ser (TGA), fMet (CAT), Asp (GTC), and Phe (GAA).

Base Sequence↗
PubMed1992

Mutagenic analysis of the coronavirus intergenic consensus sequence.

Previously, a system in which an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells was established. In the present study, a DI cDNA containing one UCUAAAC consensus sequence in the middle of the 0.3-kb-long intergenic region located between genes 6 and 7 was constructed. From this DI cDNA clone, 21 mutant DI RNAs were constructed so that each of the seven consensus sequence nucleotides was changed individually to the three alternative bases. These mutants were used to define how changes in the integrity of MHV transcription consensus sequence UCUAAAC affected mRNA transcription. Except for two mutants with the sequences UGUAAAC and UCGAAAC, all of the mutants supported efficient subgenomic DI RNA transcription. This indicated that MHV transcription regulation was sufficiently flexible to recognize altered consensus sequences. Next, these and other mutants were used to examine the leader-body fusion site on the subgenomic DI RNAs. Sequence analysis demonstrated that all subgenomic DI RNAs analyzed contained two pentanucleotide sequences; the first sequence seemed to be contributed by the leader, and the leader-body fusion most likely took place at either the first or the second nucleotide of the second sequence. This observation was not consistent with the proposed coronavirus transcription model (S. C. Baker and M. M. C. Lai, EMBO J. 9:4173-4179, 1990) which states that nucleotide mismatch can be corrected by RNA polymerase proofreading activity.

Base Sequence↗
PubMed2000

In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase.

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.

Base Sequence↗
PubMed1999

Sequence analysis of the small RNA segment of guinea pig-passaged Pichinde virus variants.

The established animal model for Lassa fever is based on the new world arenavirus Pichinde (PIC). Natural isolates of PIC virus are attenuated in guinea pigs, but serial guinea pig passage renders them extremely virulent in that host. We have compared the nucleotide sequences of the small RNA segments of two attenuated, low-passage variants of the PIC virus Munchique strain (CoAn 4763) and two virulent, high-passage derivatives. Missense mutations in the glycoprotein precursor (GPC) gene at codons GPC-119, GPC-140, and GPC-164 and the nucleoprotein gene (NP) codons NP-35 and NP-374 were most closely associated with virulence. Codon GPC-140 is predicted to represent a region of peak hydrophilicity of the glycoprotein 1 (GP1); it is conceivable that mutations at this site could influence virulence by altering B cell epitopes or virus attachment protein conformation.

Animals↗
PubMed1995

A spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in Nicotiana benthamiana.

Brome mosaic virus (BMV) is a positive-strand RNA virus with a multipartite genome that causes symptomless infection in Nicotiana benthamiana. We have isolated and characterized a strain of BMV that produced uniform vein chlorosis in systemically infected N. benthamiana. Analysis of pseudorecombinants constructed by exchanging RNA 1 and 2 and RNA 3 components between wild-type (non-symptom-inducing) and vein chlorosis-inducing strains of BMV indicated that the genetic determinant for the induction of the chlorotic phenotype is located on RNA 3. Sequence analysis of progeny RNA 3 recovered from symptomatic N. benthamiana plants revealed that vein chlorosis is due to the single nucleotide transition 887G-->887A, which changes the codon for Val-266 to Ile-266 in the movement protein gene. The mutation had no detectable effect on the accumulation of virus in either inoculated or systematically infected leaves of N. benthamiana. The vein chlorosis phenotype is the manifestation of the substitution of Ile-266 for Val-266 in the movement protein gene, since additional alterations in this region (a silent mutation, i.e., 887GUU889-->GUC, and an alteration of valine to phenylalanine, i.e., 887GUU889-->887UUU889) resulted in symptomless infections on N. benthamiana. The modulation of the symptom phenotype by the substitution of Ile-266 for Val-266 is specific for N. benthamiana, since neither movement nor the symptom phenotype in barley plants was affected.

Base Sequence↗