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At least 217 records · Page 12Linked to original sources
PubMed1993

Complex formation determines the activity of ribozymes directed against potato virus YN genomic RNA sequences.

A ribozyme was synthesized against a conserved region in the RNA-dependent RNA-polymerase encoding cistron of the important plant pathogen potato virus Y (PVY). This ribozyme was shown to cleave PVY-specific RNA-transcripts efficiently in vitro, with up to 95% of the substrate RNA being cleaved within 2 h incubation at 37 degrees C. A second ribozyme, designed with much shorter viral complementary arms in an attempt to optimize the efficiency of the cleavage reaction, surprisingly failed to cleave the substrates previously cleaved by the longer ribozyme. A much shorter PVY specific RNA-transcript of only 37 nucleotides (nt), however, was cleaved by this short ribozyme proving its ribozymic activity and indicating that the cleavage activity of the ribozyme is, in part, determined by the substrate involved. Analysis of cleavage reactions on non-denaturing polyacrylamide (PAA) gels indicated that incorrect basepairing, interfering with correct formation of the hammerhead structure, was likely to be responsible for the absence of detectable cleavage of the larger substrates by the short ribozyme.

Base Sequence↗
PubMed2002

Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon.

A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4 degrees C +/- 1 degree C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka, deltaG, and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.

Cesium↗
PubMed1988

Transcription initiation site selection and abortive initiation cycling of phage SP6 RNA polymerase.

Effects of mutations around the phage SP6 transcription initiation site on SP6 RNA polymerase's selection of initiation site were studied. In the in vitro transcription reactions, the limiting concentration of a ribonucleotide causes the SP6 RNA polymerase to stall long enough only at the positions of the limited nucleotide and dissociate from the elongation complex. As a result, a series of RNA oligomers comprises a sequencing ladder, and abortive initiation cycling products up to 6-mer are made in high yield. Precise sizing of the product RNAs from the elongation pausings determined the initiation site of each mutant. When the wild-type +1 G is changed to C or A without change in the upstream sequence including TATA from -4 to -1, transcription still starts only at the +1 site. But, the mutant containing TATCC from -4 to +1 C. We propose that the phage SP6 RNA polymerase selects the initiation site precisely at a certain distance from a direct contact point in the upstream promoter sequence, regardless of the species of initiating nucleotide. It is also suggested that the sequence-dependent perturbations of DNA helical structure, for example D to B form, may shift the initiation site.

Bacteriophages↗
PubMed2001

Differential roles of viral RNA and cRNA in functional modulation of the influenza virus RNA polymerase.

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multifunctional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA binding, capped RNA cleavage, viral model RNA binding, model RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirus-infected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vRNA but not of cRNA, indicating that the vRNA functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates.

Baculoviridae↗
PubMed1988

A sequence motif in many polymerases.

A 15-residue sequence motif has been found in many polymerases from various species and involving DNA and RNA dependence and product. The motif is characterized by a Tyr-Gly-Asp-(Thr)-Asp core flanked by hydrophobic spans five residues in length. An mRNA maturase segment is also suggested to display the motif pattern. The aspartates may be important in polymerase function by acting directly in catalysis and/or by binding magnesium.

Amino Acid Sequence↗
PubMed2003

Mutations in the amino terminus of ANKH in two US families with calcium pyrophosphate dihydrate crystal deposition disease.

OBJECTIVE: To analyze ANKH in families with calcium pyrophosphate dihydrate crystal deposition disease (CPPD) for disease-causing mutations. METHODS: Two US families (one of British ancestry and the other of German/Swiss ancestry) with autosomal-dominant CPPD, whose disease phenotypes were found to be linked to chromosome 5p15.1 (locus symbol CCAL2), were screened by direct sequencing for mutations in ANKH, a gene in the CCAL2 candidate interval that has been shown to harbor mutations in other families with CPPD. Observed sequence variants were confirmed by antisense sequencing, and expression of the mutant allele was verified by reverse transcriptase-polymerase chain reaction amplification of messenger RNA followed by direct sequencing. RESULTS: The two US families displayed the same mutation at position 5 of the ANKH gene product (P5T). All affected members were heterozygous for the P-to-T variant, and the mutation was not seen in 204 control alleles. The two families displayed distinct disease haplotypes, suggesting that they were unrelated to each other. CONCLUSION: These observations represent the fourth and fifth families with heritable CPPD whose disease phenotypes are linked to the CCAL2 locus and who have missense mutations in the amino terminus of ANKH. This same position (P5) was the site of a missense mutation in an Argentine family of northern Italian ancestry; however, the sequence variant in that family generated a P5L mutation. The distinct disease haplotypes among the 3 families with P5 mutations suggest that the mutations arose independently and that the evolutionarily conserved P5 position of ANKH may represent a hot spot for mutation in families with autosomal-dominant CPPD.

Chondrocalcinosis↗
PubMed1999

Similarity relations of DNA and RNA polymerases investigated by the principal component analysis of amino acid sequences.

The principal component analysis based on the physicochemical properties of amino acid residues is applied to DNA and RNA polymerases to assign the sequence motifs for the polymerization activities of these proteins. After the reconfirmation of the sequence motifs of families A and B of DNA polymerases indicated previously, it elucidates the sequence motifs for the polymerization activity of DNA polymerase III (family C) by the similarity to the polymerization center of multimeric DNA dependent RNA polymerases. This identification proceeds to clarify the sequence motifs for polymerization activities of primases; eukaryotic and archaebacterial primases carry motifs similar to those of family C, while the motifs of eubacterial primase fall into the category of the motifs in family B DNA polymerases such as alpha, delta, epsilon and II. This finding means that DNA dependent RNA polymerases are also divided into groups corresponding to three families, A, B and C, because the monomeric DNA dependent RNA polymerases in phages are reconfirmed to carry sequence motifs similar to those of family A DNA polymerases. Furthermore, the three families of polymerization motifs are found to fall within the variation range of polymerization motifs displayed by many RNA dependent RNA polymerases, suggesting a close evolutionary relation between them. The sequence motifs for polymerization activities of reverse transcriptase and telomerase seem to be the intermediate between family A DNA polymerase and some RNA dependent RNA polymerases, e.g., from Leviviridae. On the contrary, the sequence fragments similar to the nucleotidyltransferase superfamily including DNA polymerase beta are not found in any RNA dependent RNA polymerase, suggesting their other lineage of polymerization motifs.

Amino Acid Sequence↗
PubMed1982

Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor.

Heterogeneous nuclear protein complexes (hnRNP) containing the precursor RNA from the adenovirus early region 2 were analysed to determine the specificity of protein-RNA interaction. RNA precursor sequences were present in isolated hnRNP complexes and endogenous 30S particles. At least 20-40 bases long fragments were protected when RNase A was used to remove unprotected RNA sequences in hnRNA complexes. Similarly around 40 bases of RNA were protected in 30S particles. These sequences represent discrete regions of the adenovirus genome. Especially sequences complementary to the EcoRI-F fragment encoding the first leader and the major intron for the DNA binding protein (DBP) RNA precursor, were analysed in detail. Tentatively, sequences resistant to RNase A were located in the middle of the intron and at the splice-donor junction of the first leader of the DBP precursor RNA. The same sequences were identified irrespective whether hnRNP complexes or 30S particles were used suggesting that 30S particles originate from hnRNP complexes. A 38.000 dalton protein appears to be in direct contact with RNA sequences complementary to the EcoRI-F fragment.

Adenoviruses, Human↗
PubMed1998

Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis.

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.

Base Sequence↗
PubMed1994

Fidelity of nucleic acid amplification with avian myeloblastosis virus reverse transcriptase and T7 RNA polymerase.

The Nucleic Acid Sequence-Based Amplification (NASBA) process involves alternate steps of DNA synthesis from an RNA template and RNA synthesis from a DNA template, using avian myeloblastosis virus (AMV) reverse transcriptase and T7 RNA polymerase, respectively. The overall fidelity of the amplification process was determined by sequence analysis of cloned DNA products of NASBA reactions. An error frequency of less than 0.3% was observed in cloned DNA products from two different segments of the HIV-1 gag gene. Partial substitution of GTP with ITP in the NASBA reaction did not significantly change the fidelity of the process. An error rate of 2 x 10(-4) was calculated for the combined effects of both polymerases.

Avian Myeloblastosis Virus↗
PubMedDate not supplied

A comparison of RNA with DNA in template-directed synthesis.

Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

Base Pairing↗
PubMed1988

The human U1 snRNA promoter and enhancer do not direct synthesis of messenger RNA.

We examined the ability of the 5' flanking region sequences of a human U1 RNA gene to direct synthesis of functional mRNA. When fused to chloramphenicol acetyltransferase (CAT) coding region sequences, the upstream sequences of the U1 gene were able to stimulate the synthesis of functional CAT mRNA in 293 cells but not in HeLa cells. Most of the polyadenylated CAT mRNA in 293 cells originated from cryptic promoters in the upstream U1 sequences, but nearly all of the CAT-specific RNA originating at position +1 (relative to the U1 gene promoter) was non-polyadenylated; this confirmed that the bona-fide U1 gene promoter was unable to direct efficient synthesis of poly-A+ mRNA. Our results demonstrate that the snRNA gene promoter and enhancer elements, although very efficient in transcription of snRNAs, are unable to direct transcription of polyadenylated mRNAs. However, other sequences in the 5' flanking region of the human U1 gene can activate transcription of functional mRNA, with 5' ends upstream of the normal transcription start site.

Acetyltransferases↗
PubMed1992

Nucleotide sequence of prothrombin gene in abnormal prothrombin-producing hepatocellular carcinoma cell lines.

A protein induced by vitamin K absence or antagonist II, PIVKA-II is synthesized in the liver and possesses a structure similar to prothrombin except that ten glutamic acid residues in amino-terminal Gla domain are not completely gamma-carboxylated and are functionally inactive. This protein can be detected in the plasma of patients with hepatocellular carcinoma (HCC) and used as a new tumor marker. To analyze the mechanism of PIVKA-II production in HCC tissue, the prothrombin gene of PIVKA-II-secreting HCC cell lines was sequenced to detect the mutation in the Gla domain and carboxylase recognition site of leader sequence located on exons I and II that may cause the inhibition of carboxylation. Exons I and II and donor and acceptor site of intron I of the prothrombin gene in two HCC cell lines, PLC/PRF/5 and huH-2, were analyzed by polymerase chain reaction (PCR), and the product was sequenced directly. In addition, RNA samples of these cell lines were used for complementary DNA synthesis, followed by PCR and sequencing. The nucleotide sequences of the Gla domain in both HCC cell lines were conserved. One nucleotide change was detected at nt.554 (adenine to guanine), but this did not influence the amino acid sequence. Splicing sites between exons I and II, the leader sequence of the precursor prothrombin, and protease target sites also were conserved as the reported prothrombin gene, and mutations reported for other des-gamma-carboxy coagulation factors were not detected. These results also were confirmed by DNA analysis of seven human fresh-frozen samples (three PIVKA-II-positive HCC samples and four control specimens). The mechanism of PIVKA-II production in HCC is still unclear, but it is not caused by mutation in the prothrombin gene.

Amino Acid Sequence↗
PubMed1994

Comparison of the polymerase region of small round structured virus strains previously classified in three antigenic types by solid-phase immune electron microscopy.

We have used a reverse transcription-polymerase chain reaction with nested sets of primers to determine the nucleotide sequences of a 166 base pair segment of the RNA polymerase region of seven strains of small round structured viruses (SRSVs) from the United Kingdom. These SRSV strains were previously classified by solid-phase immune electron microscopy into three antigenic types--UK2, UK3 and UK4, which are comparable to the prototype strains Norwalk virus, Hawaii agent, and Snow Mountain agents, respectively. Based on their sequences, the seven strains from the United Kingdom could be divided into two groups. The first group included two strains of the UK2 type along with Norwalk virus and Southampton virus and the second group included three strains of UK3 and two strains of UK4 types. Viruses in the first group showed 75.3%-77.1% nucleotide and 89.1%-94.6% amino acid identity with Norwalk virus while those of the second group showed 60.8%-63.3% nucleotide and 67.3%-69.1% amino acid identity. Nucleotide and amino acid identity within the second group ranged between 91.6%-99.4% and 96.4%-100%, respectively. These results suggest that the SRSVs antigenically related with Norwalk virus, Hawaii agent, and Snow Mountain agent, can be classified into two genotypes on the basis of their sequences in the RNA polymerase region.

Adult↗
PubMed2000

Determination of the rpoB gene sequences of Bartonella henselae and Bartonella quintana for phylogenic analysis.

Using the Genome Walker procedure, which allows PCR amplification of genomic DNA using a single gene-specific primer and direct automated sequencing methodology, we obtained the nucleotide sequence of the RNA polymerase beta subunit (rpoB) from Bartonella henselae and Bartonella quintana. A phylogenetic tree constructed from these data and other rpoB sequences available in GenBank is, in part, consistent with those previously derived from 16S rRNA gene sequences and confirms the position of Bartonella within the alpha subdivision of Proteobacteria. In fact, this analysis showed that rpoB data are similar to 16S rRNA data for the alpha, beta and gamma subdivisions of Proteobacteria. In contrast, concerning other bacteria included in our study, the topologies of phylogenetic trees were different. Based on the bootstrap values derived from rpoB phylogenic analysis, we believe that this molecule should contribute to better understanding the evolutionary process.

Amino Acid Sequence↗
PubMed1996

Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes.

Efficacy and sequence specificity are two major requirements in the use of antisense nucleic acids and ribozymes. For long-chain complementary RNA sequences (>30 nt), effects in living cells are correlated with the association rate of the complementary RNA in vitro, but not with the stability of the formed double strand. Thus, sequence selectivity of complementary RNA has to be defined as fast versus slow annealing with the appropriate target or non-target sequences, respectively. In this work, we performed a systematic kinetic analysis to evaluate the selectivity of bcr-abl-directed antisense RNA and hammerhead ribozymes with a length of the complementary sequences of between 20 and 80 bases. By kinetic in vitro selection, we identified oligomeric as well as long-chain complementary RNA that annealed at least tenfold faster with the bcr-abl sequence in comparison with either of the wild-type sequences bcr or abl, respectively. In the presence of selected oligodeoxynucleotide sequences and RNase H, the bcr-abl transcript was specifically hydrolysed out of a mixture containing abl and bcr sequences as well. Hammerhead ribozymes were designed such that binding with their target was facilitated either via helix I or helix III-forming antisense arms but not both. Further, cleavage and binding occurred on opposite sides of the bcr-abl fusion point. Target selectivity was found for a ribozyme that annealed fast via abl sequences and cleaved within the bcr portion of bcr-abl RNA. Kinetic probing and calculations of the local folding potential indicate that the bcr-abl fusion point sequences are not easily accessible for complementary nucleic acids. This study supports the need for more detailed structural investigations of the bcr-abl fusion sequence and forms a more rational basis for the therapeutic use of nucleic acid inhibitors of the aberrant bcr-abl gene expression in Philadelphia chromosome-positive cells.

Base Sequence↗