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At least 1,693 records · Page 94Linked to original sources
PubMed1998

Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation.

The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human beta-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless beta-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes.

Animals↗
PubMed1984

Expression of unspliced NS1 mRNA, spliced NS2 mRNA, and a spliced chimera mRNA from cloned influenza virus NS DNA in an SV40 vector.

Influenza virus gene 8 codes for two nonstructural proteins (NS1 and NS2) which are translated, respectively, from a colinear and an interrupted mRNA. To investigate the mechanism of transcription processing by splicing, cloned full-length NS DNA was inserted into the late region of an SV40 vector. In this manner, transcription of NS RNA from the recombinant initiates and terminates using SV40 control sequences. RNA mapping and sequence analysis showed that the RNA transcripts are processed to produce both the spliced and unspliced NS mRNAs in approximately equal abundance. The junction sequence of the spliced mRNA is identical to that of NS2 mRNA found in influenza virus-infected cells. In addition, another mRNA species was found to contain a chimera splice between SV40 and NS sequences. Both NS1 and NS2 polypeptides are produced in the recombinant-infected cells as predicted from sequence analysis. These studies establish that during influenza virus infections processing of the NS1 mRNA transcript undergoes a mechanism of splicing similar to that occurring with DNA-directed RNA transcription.

Amino Acid Sequence↗
PubMedDate not supplied

Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA.

We sequenced a 173-nucleotide fragment of the small double-stranded viruslike RNA of Cryptosporidium parvum isolates from 23 calves and 38 humans. Sequence diversity was detected at 17 sites. Isolates from the same outbreak had identical double-stranded RNA sequences, suggesting that this technique may be useful for tracking Cryptosporidium infection sources.

Animals↗
PubMed2006

MODOMICS: a database of RNA modification pathways.

MODOMICS is the first comprehensive database resource for systems biology of RNA modification. It integrates information about the chemical structure of modified nucleosides, their localization in RNA sequences, pathways of their biosynthesis and enzymes that carry out the respective reactions. MODOMICS also provides literature information, and links to other databases, including the available protein sequence and structure data. The current list of modifications and pathways is comprehensive, while the dataset of enzymes is limited to Escherichia coli and Saccharomyces cerevisiae and sequence alignments are presented only for tRNAs from these organisms. RNAs and enzymes from other organisms will be included in the near future. MODOMICS can be queried by the type of nucleoside (e.g. A, G, C, U, I, m1A, nm5s2U, etc.), type of RNA, position of a particular nucleoside, type of reaction (e.g. methylation, thiolation, deamination, etc.) and name or sequence of an enzyme of interest. Options for data presentation include graphs of pathways involving the query nucleoside, multiple sequence alignments of RNA sequences and tabular forms with enzyme and literature data. The contents of MODOMICS can be accessed through the World Wide Web at http://genesilico.pl/modomics/.

Base Sequence↗
PubMed1993

Nucleotide sequence of the primer RNA for DNA replication of filamentous bacteriophages.

We determined the nucleotide sequence of RNA synthesized in vitro by Escherichia coli RNA polymerase at the complementary-strand replication origin on the single-stranded viral DNA of bacteriophages f1 and IKe (ori-RNA) by using chain-terminating ribonucleoside triphosphate analogs. The results indicated that the start site of f1 ori-RNA synthesis is 20 nucleotides downstream from the site previously reported (K. Geider, E. Beck, and H. Schaller, Proc. Natl. Acad. Sci. USA 75:645-649, 1978) and that the RNA sequence [(5')pppAGGGCGAUGGCCCACUACGU-OH(3')] is complementary to the f1 DNA sequence from nucleotides 5736 to 5717, with minor heterogeneity at the 3' end. IKe ori-RNA had a sequence identical to that of f1 ori-RNA, except for a single base substitution, and IKe RNA was complementary to a region of IKe DNA (from nucleotides 6441 to 6422) that was homologous to the f1 sequence. Phenotypes and ori-RNA sequences in the relevant region of the genome of f1 deletion mutants were consistent with the presently determined sequence of ori-RNA. A possibility that ori-RNA synthesis is initiated by a mechanism similar to that for general transcription is suggested as a result of the new assignment of the ori-RNA start site. The double-origin plasmid assay of minus-strand origin activity, a sensitive in vivo method for detecting cis-acting elements for the initiation of DNA replication on a single-stranded DNA template, is described.

Base Sequence↗
PubMed1997

Inhibition of Moloney murine leukaemia virus by a retroviral vector, LNL6, carrying ribozymes targeted to the 5' non-coding sequence.

The packaging region psi is the RNA sequence which directs the inclusion of genomic viral RNA into virions. As such it represents an apparently accessible site for ribozyme action. Ribozymes directed against the psi site of Moloney murine leukaemia virus (MoMLV) were delivered to target cells using a related retroviral vector, LNL6. LNL6 is a vector predominantly derived from MoMLV and, like all retroviruses, requires the psi region to be packaged. By exploiting the heterogeneity between nucleotide sequences of the MoMLV target and LNL6 vector in the psi packaging region, two ribozymes were designed and shown to selectively cleave the target but not the vector sequence. Clonally derived cell lines expressing ribozymes showed inhibition of virus replication. These results show the utility of catalytic RNA as specific antiviral agents.

3T3 Cells↗
PubMed1996

Comparative analysis of tertiary structure elements in signal recognition particle RNA.

BACKGROUND: The signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote co-translational translocation of proteins across biological membranes. We have used comparative analysis of a large number of bacterial, archaeal, and eukaryotic SRP RNA sequences to derive shared tertiary SRP RNA structure elements. RESULTS: A representative three-dimensional model of the human SRP RNA is shown that includes single-stranded intrahelical and interhelical RNA loops and incorporates data from enzymatic and chemical modification, electron microscopy, and site-directed mutagenesis. Properties of the SRP RNA model are an overall extended dumbbell-shaped structure (260 A x 70 A) with a pseudoknot in the small SRP domain (a pairing of 12-UGGC-15 with 33-GCUA-36), and a tertiary interaction in the large SRP domain (198-GA-199 with 232-GU-233). CONCLUSIONS: The RNA 'knuckle' formed in helix 8 of SRP RNA appears to constitute the binding site for protein SRP54 or its bacterial equivalent, protein P48. A dynamic property of this feature may explain the hierarchial assembly of proteins SRP19 and SRP54 in the large SRP domain. Furthermore, the human SRP RNA model serves as a framework to understand details of the structure and function of SRP in all organisms and is presented to stimulate further experimentation in this area.

Animals↗
PubMed1989

srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda).

A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent.

Animals↗
PubMed1978

Nucleotide sequence of Xenopus borealis oocyte 5S DNA: comparison of sequences that flank several related eucaryotic genes.

Genomic Xenopus borealis oocyte-specific 5S DNA (Xbo) contains clusters of 5S rRNA genes. The number of genes varies among clusters, and the distance between genes within a cluster is about 80 nucleotides. The spacer DNA between gene clusters is AT-rich and heterogeneous in length due in part to variable numbers of a tandemly repeated 21 nucleotide sequence. A cloned fragment of Xbo 5S DNA (Xbo1) containing three 5S rRNA genes has been sequenced. The sequences of Xbo1 genes 1 and 2 are very similar to the dominant 5S RNA sequence, whereas 15 of the 120 residues in the third gene are different. The sequence of gene 3 is as different from the dominant gene sequence as the X. laevis pseudogene is from the 5S RNA gene. Sequence analysis of genomic DNA shows that gene 3 is an abundant component of the multigene family. All three genes are transcribed when added to an extract of X. laevis oocyte nuclei, and a fragment of Xbo1 lacking the AT-rich spacer DNA and the 5' end of the first gene supports transcription of genes 2 and 3 in this in vitro system. Thus the 80 nucleotides preceding each 5S gene are sufficient for promoter function. Nucleic acid sequences preceding several eucaryotic genes that are transcribed by RNA polymerase III were analyzed and the following common features were found: a purine-rich region; at least one direct repeat; the absence of dyad symmetry; transcription beginning with a purine; a pyrimidine residue immediately preceding the first nucleotide of the gene; and the oligonucleotides AAAAG, AGAAG and GAC, located approximately 15, 25 and 35 nucleotides, respectively, before the start of transcription. The 10 base pair (bp) spacing between the homologous oligonucleotides is that expected for a recognition signal on one face of a DNA double helix. The extensive sequence differences between most of the spacers that precedes these genes make the three conserved oligonucleotides more striking. Parts of the 5' flanking regions of the three Xbo1 gene (-12 to -40), which include the conserved oligonucleotides, are identical. In contrast, 7 of the first 11 nucleotides that precede the third 5S RNA gene in Xbo1 differ from those that precede the first gene. The sequences following the X. borealis oocyte and somatic 5S genes are identical in 12 of the first 14 residues and contain two or more T clusters, as does the corresponding region of X. laevis oocyte 5S DNA. The 3' sequences of the Xenopus 5S rRNA genes and several other eucaryotic genes contain features in common with procaryotic transcription termination sites. The 3' end of the gene is GC-rich and contains a dyad symmetry. Termination occurs in an AT-rich region containing one or more T clusters on the noncoding strand.

Animals↗
PubMed2004

Pattern of 4-thiouridine-induced cross-linking in 16S ribosomal RNA in the Escherichia coli 30S subunit.

The locations of RNA-RNA cross-links in 16S rRNA were determined after in vivo incorporation of 4-thiouridine (s(4)U) into RNA in a strain of Escherichia coli deficient in pyrimidine synthesis and irradiation at >320 nm. This was done as an effort to find RNA cross-links different from UVB-induced cross-links that would be valuable for monitoring the 30S subunit in functional complexes. Cross-linked 16S rRNA was separated on the basis of loop size, and cross-linking sites were identified by reverse transcription, RNase H cleavage, and RNA sequencing. A limited number of RNA-RNA cross-links in nine regions were observed. In five regions-s(4)U562 x C879-U884, s(4)U793 x A1519, s(4)U1189 x U1060-G1064, s(4)U1183 x A1092, and s(4)U991 x C1210-U1212-the s(4)U-induced cross-links are similar to UVB-induced cross-links observed previously. In four other regions-s(4)U960 x A1225, s(4)U820 x G570, s(4)U367 x A55-U56, and s(4)U239 x A120-the s(4)U-induced cross-links are different from UVB-induced cross-links. The pattern of cross-linking is not limited by the distribution of s(4)U, because there are at least 112 s(4)U substitution sites in the 16S rRNA. The relatively small number of s(4)U-mediated cross-links is probably determined by the organization of the RNA in the 30S subunit, which allows RNA conformational flexibility needed for cross-link formation in just a limited region.

Base Sequence↗
PubMed1999

Subgenomic mRNA regulation by a distal RNA element in a (+)-strand RNA virus.

Subgenomic (sg) mRNAs are synthesized by (+)-strand RNA viruses to allow for efficient translation of products encoded 3' in their genomes. This strategy also provides a means for regulating the expression of such products via modulation of sg mRNA accumulation. We have studied the mechanism by which sg mRNAs levels are controlled in tomato bushy stunt virus, a small (+)-strand RNA virus which synthesizes two sg mRNAs during infections. Neither the viral capsid nor movement proteins were found to play any significant role in modulating the accumulation levels of either sg mRNA. Deletion analysis did, however, identify a 12-nt-long RNA sequence located approximately 1,000 nt upstream from the site of initiation of sg mRNA2 synthesis that was required specifically for accumulation of sg mRNA2. Further analysis revealed a potential base-pairing interaction between this sequence and a sequence located just 5' to the site of initiation for sg mRNA2 synthesis. Mutant genomes in which this interaction was either disrupted or maintained were analyzed and the results indicated a positive correlation between the predicted stability of the base-pairing interaction and the efficiency of sg mRNA2 accumulation. The functional significance of the long-distance interaction was further supported by phylogenetic sequence analysis which revealed conservation of base-pairing interactions of similar stability and relative position in the genomes of different tombusviruses. It is proposed that the upstream sequence represents a cis-acting RNA element which facilitates sg mRNA accumulation by promoting efficient synthesis of sg mRNA2 via a long-distance RNA-RNA interaction.

Amino Acid Sequence↗
PubMed2004

Paradigms for computational nucleic acid design.

The design of DNA and RNA sequences is critical for many endeavors, from DNA nanotechnology, to PCR-based applications, to DNA hybridization arrays. Results in the literature rely on a wide variety of design criteria adapted to the particular requirements of each application. Using an extensively studied thermodynamic model, we perform a detailed study of several criteria for designing sequences intended to adopt a target secondary structure. We conclude that superior design methods should explicitly implement both a positive design paradigm (optimize affinity for the target structure) and a negative design paradigm (optimize specificity for the target structure). The commonly used approaches of sequence symmetry minimization and minimum free-energy satisfaction primarily implement negative design and can be strengthened by introducing a positive design component. Surprisingly, our findings hold for a wide range of secondary structures and are robust to modest perturbation of the thermodynamic parameters used for evaluating sequence quality, suggesting the feasibility and ongoing utility of a unified approach to nucleic acid design as parameter sets are refined further. Finally, we observe that designing for thermodynamic stability does not determine folding kinetics, emphasizing the opportunity for extending design criteria to target kinetic features of the energy landscape.

Algorithms↗
PubMed1995

Plasmodium: a quantitative molecular assay for detection of sporogonic-stage malaria parasites.

We present a molecular assay to detect malaria parasites during sporogonic development in the mosquito host. Specific primers for Plasmodium-specific small-subunit ribosomal RNA sequences not present in mosquito RNA were used in a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A synthetic RNA quantitative competitor was made which included targets for two primers and a target sequence for a hybridization probe which is also present in the natural parasite ribosome. The heterobifunctional Tth polymerase was used to carry out both reverse transcription and DNA-dependent polymerase chain reaction in a single reaction tube. Ookinetes and sporozoites, the stages from the beginning and end of sporogonic development, respectively, were both recognized in the assay. The assay was calibrated for quantitation of sporozoites by making a standard curve with counted sporozoites. The linear range of the calibrated assay allowed accurate quantitation of parasite number over at least two orders of magnitude, from 10 to 1000 sporozoites, in each RT-PCR reaction.

Aedes↗
PubMed1989

The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein.

A method of affinity purification of a regulatory protein that binds specific RNA sequences is described. RNAs containing the regulatory sequences are transcribed in vitro from oligonucleotide templates, biotinylated, and incubated with unfractionated cytosol. Specific RNA-protein complexes are bound in solution to avidin, and the resulting complex is bound to biotin-agarose beads. The cytosolic binding protein is released from the RNA in high salt, and a second round of purification yields an essentially homogeneous protein. Using this method, we have identified the protein in human liver that binds iron-responsive RNA regulatory sequences. Iron-responsive elements (IREs) are RNA stem-loops present in the mRNAs encoding ferritin and the transferrin receptor. IREs form the basis for the translational regulation of ferritin gene expression and the regulation of transferrin receptor mRNA degradation rates. The IRE binding protein purified by this technique migrates as a 90-kDa polypeptide on SDS/PAGE. The interaction of the purified protein with IRE-containing RNAs can be detected by gel-mobility shift assays or by covalent crosslinking induced by UV irradiation.

Base Sequence↗
PubMed1995

When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells.

It appears that no organism is immune to the effects of nonsense codons on mRNA abundance. The study of how nonsense codons alter RNA metabolism is still at an early stage, and our current understanding derives more from incidental vignettes than from experimental undertakings that address molecular mechanisms. Challenges for the future include identifying the gene products and RNA sequences that function in nonsense mediated RNA loss, resolving the cause and consequences of there apparently being more than one cellular site and mechanism for nonsense-mediated RNA loss, and understanding how these sites and mechanisms are related to both constitutive and specialized pathways of pre-mRNA processing and mRNA decay.

Animals↗
PubMed1985

Characterization of leader-related small RNAs in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription.

Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNAs containing an identical stretch of leader RNA sequences at the 5'-end of each RNA. The leader-coding sequences at the 5'-end of genomic RNA are at least 72 nucleotides in length and are joined to the viral mRNAs by a unique mechanism. Utilizing a leader-specific cDNA probe, we have detected several free leader RNA species ranging from 70 to 82 nucleotides in length. The predominant leader RNA was approximately 75 nucleotides. In addition, larger distinct leader-containing RNAs were also detected ranging from 130 to 250 nucleotides in length. The 70-82-nucleotide leader-related RNAs were present in both the cytosol and membrane fractions of infected cells. They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA. These data suggest that the leader RNAs were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template. We have also identified a temperature-sensitive mutant, which synthesizes only leader RNA but not mRNAs at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNAs. These data support the leader-primed mechanism for coronavirus transcription and suggest that one or more free leader RNAs are used as primers of mRNA synthesis.

Animals↗
PubMed2001

Mutations in the NS3 gene and 3'-NCR of Japanese encephalitis virus isolated from an unconventional ecosystem and implications for natural attenuation of the virus.

The T1P1 strain of Japanese encephalitis (JE) virus was recently isolated from paddy-free Liu-Chiu Islet in which natural JE antibody has been prevalent. In mouse neuroblastoma-derived Neuro-2a cells, T1P1 appeared significantly lower in virus productivity than another local isolate, CH1392. It implied that this new isolate possesses a characteristic viral replication pattern other than that of CH1392. T1P1 has also shown lower neurovirulence, which was reflected by a significantly higher LD(50) (2.44 x 10(6) PFU) than CH1392 (2.87 x 10(2) PFU). In comparison of the full-length RNA sequences between T1P1 and CH1392, a total of 7 nucleotides, including 1 in preM/M and 2 each in NS3, NS5, and the 3'-end noncoding region (NCR), appeared different. Of them, only the changes in NS3 (position 325, T for CH1392, A for T1P1; and position 364, G for CH1392 and A for T1P1) resulted in substitutions of deduced amino acids. There were two additional nucleotide changes appearing in the 3'-NCR. The amino acids 109 Phe and 122 Glu in NS3 of CH1392 were substituted by Ile and Lys, respectively, in T1P1. The unique growth properties and low virulence of T1P1 presented in this report were likely related to abnormal enzymatic activity due to mutations of the NS3 gene (especially position 364) and possibly to the mutations in the 3'-NCR. The natural attenuation of T1P1 that has been circulating in paddy-free Liu-Chiu Islet may account for the absence of clinical JE cases in past years.

3' Untranslated Regions↗
PubMed1978

Nucleotide sequence heterogeneity of an RNA phage population.

The nucleotide sequence of 32P-RNA from Q beta phage clones was sampled by two-dimensional polyacrylamide gel electrophoresis of the RNAase T1-resistant oligonucleotides (T1 fingerprinting). About 15% of the clones derived from a multiply passaged Q beta population showed fingerprint patterns which deviated from that of the RNA from the total population. All deviations examined could be attributed to one and, less frequently, to two or more nucleotide transitions. Since the fingerprinting technique allows the analysis of only about 10% of the RNA sequence, we estimate that each viable phage genome in a multiply passaged population differs in one to two positions from the "average" sequence of the parental population. Several deviant clones were tested by growth competition against a "wildtype" population, after 10-20 generations, the resulting phage showed the "wild-type" T1 fingerprint pattern. We propose that a Q beta phage population is in a dynamic equilibrium, with viable mutants arising at a high rate (Batschelet, Domingo and Weissmann, 1976; Domingo, Flavell and Weissmann, 1976) on the one hand, and being strongly selected against on the other. The genome of Q beta phage cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.

Base Sequence↗