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At least 397 records · Page 22Linked to original sources
PubMed1988

Identification of a repeated sequence in the genome of the sea urchin which is transcribed by RNA polymerase III and contains the features of a retroposon.

A repeated sequence element which is located about 200 nucleotides upstream from the protein-coding portion of the muscle actin gene (probably within a large 5' intron) in the genome of the sea urchin, Strongylocentrotus purpuratus has been characterized, and shown to contain the sequence features which indicate that it has been transposed by means of an RNA intermediate. This retroposon-like sequence, SURF1-1, is a member of a family which is dispersed and repeated about 800 times in the genome, referred to as SURF1 (sea urchin retroposon family 1). In vitro transcription of this sequence by RNA polymerase III defines a 300 nucleotide transcription unit which is bounded by a short direct repeated sequence. The 3' end of this unit contains a simple 21 nucleotide A+T-rich sequence characteristic of retroponons, and a consensus B box portion of an internal RNA polymerase III promotor is located 60 to 80 nucleotides downstream from the two sites of transcription initiation. This sequence also contains a 40 nucleotide region that is related to several tRNA sequences (containing the B box), and a 79 nucleotide sequence which is homologous to a repeated sequence previously shown to be present within the 3' untranslated portions of the Spec1 and Spec2 mRNAs of this species (1). Analysis of transcripts of this sequence family in RNA from several embryonic stages indicates that its expression is highest at 11 hours postfertilization (about 128 cells) and drops as development proceeds. Furthermore, most or all, transcription of this sequence family in nuclei isolated from 11 hour embryos is by RNA polymerase III, and is from the same strand which is transcribed in vitro.

Actins↗
PubMed1993

Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis.

Control of tuberculosis is threatened by widespread emergence of drug resistance in Mycobacterium tuberculosis. Understanding the molecular basis of resistance might lead to development of novel rapid methods for diagnosing drug resistance. We set out to determine the molecular basis of resistance to rifampicin, a major component of multidrug regimens used for treating tuberculosis. Resistance to rifampicin involves alterations of RNA polymerase. The gene that encodes the RNA polymerase subunit beta (rpoB) was cloned. Sequence information from this gene was used to design primers for direct amplification and sequencing of a 411 bp rpoB fragment from 122 isolates of M tuberculosis. Mutations involving 8 conserved aminoacids were identified in 64 of 66 rifampicin-resistant isolates of diverse geographical origin, but in none of 56 sensitive isolates. All mutations were clustered within a region of 23 aminoacids. Thus, substitution of a limited number of highly conserved aminoacids encoded by the rpoB gene appears to be the molecular mechanism responsible for "single step" high-level resistance to rifampicin in M tuberculosis. This information was used to develop a strategy (polymerase chain reaction-single-strand conformation polymorphism) that allowed efficient detection of all known rifampicin-resistant mutants. These findings provide the basis for rapid detection of rifampicin resistance, a marker of multidrug-resistant tuberculosis.

Base Sequence↗
PubMed1994

Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus.

The complete nucleotide sequence of the genomic RNA of bamboo mosaic virus (BaMV) was determined by sequencing a set of overlapping cDNA clones and by direct sequencing of the viral RNA. The RNA genome of BaMV is 6366 nucleotides long [excluding 3'poly(A) tail] and contains six open reading frames (ORFs 1 to 6) coding for polypeptides with M(r) values of 155K, 28K, 13K, 6K, 25K and 14K, respectively. The genome organization and sizes of the encoded proteins are very similar to those of other potexviruses which have been sequenced except that ORF 6 lies completely within ORF 1. The first five putative proteins of the BaMV genome show identities ranging between 44 to 59%, 26 to 49%, 30 to 53%, 15 to 35% and 20 to 30%, respectively, to the corresponding ORFs of other members of the potexvirus group. However the putative product ORF 6 shows no significant similarity to those of other potexvirus ORF products.

Amino Acid Sequence↗
PubMed1994

Geographic distribution and evolution of yellow fever viruses based on direct sequencing of genomic cDNA fragments.

We have compared the nucleotide sequence of an envelope protein gene fragment encoding amino acids 291 to 406 of 22 yellow fever (YF) virus strains of diverse geographic and host origins isolated over a 63 year time span. The nucleotide fragment of viral RNA was examined by direct sequencing of a PCR product derived from complementary DNA. Alignment with the prototype Asibi strain sequence showed divergence of 0 to 21.5% corresponding to a maximum of 5.2% divergence in the amino acid sequence. Taking 10% nucleotide divergence as a cut-off point, the 22 YF virus strains fell into three topotypes which corresponded to different geographical areas, namely West Africa, Central-East Africa, and South America. Two subgroups were defined in West Africa, a genotypic group circulating in the sylvatic zone of the western part of Africa, from western Ivory Coast-Mali to Senegal, and a group responsible for large outbreaks from eastern Ivory Coast-Burkina Faso to Cameroon. Strains from Central-East Africa showed a low ratio of transition:transversion of about 1 instead of 8 to 10 for other strains, when their nucleotide sequences were compared with those of other African strains. This may reflect a more distant relationship between the former strains and the others. No change was observed in the highly conserved amino acid domain encompassing the TGD sequence, an important determinant of flavivirus tropism and pathogenesis. Our results support earlier observations on the genetic relationships between YF isolates established by T1 oligonucleotide fingerprinting and offer a useful tool for the understanding of YF virus distribution and evolution.

Amino Acid Sequence↗
PubMed1999

Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer.

The complete nucleotide sequence was determined for the putative RNA polymerase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which differed from the related strain, vulgare, by 51 positions in its nucleotide sequence and 6 residues in its amino acid sequence. Three segments of this 183K protein, each containing the sequence motif of methyltransferase (M), helicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escherichia coli as fusion proteins with hexahistidine tags, and domain-specific antibodies were raised against purified His-tagged M and P polypeptides. By immunoaffinity purification, a template-specific RNA-dependent RNA polymerase containing a heterodimer of the full-length 183K and 126K (an amino-terminal-proximal portion of the 183K protein) viral proteins was isolated. We propose that the TMV RNA polymerase for minus-strand RNA synthesis is composed of one molecule each of the 183- and 126-kDa proteins, possibly together with two or more host proteins.

Base Sequence↗
PubMed1995

Crystallisation of RNA-protein complexes. II. The application of protein engineering for crystallisation of the U1A protein-RNA complex.

The hairpin is one of the most commonly found structural motifs of RNA and is often a binding site for proteins. Crystallisation of U1A spliceosomal protein bound to a RNA hairpin, its natural binding site on U1snRNA, is described. RNA oligonucleotides were synthesised either chemically or by in vitro transcription using T7 RNA polymerase and purified to homogeneity by gel electrophoresis. Crystallisation trials with the wild-type protein sequence and RNA hairpins containing various stem sequences and overhanging nucleotides only resulted in a cubic crystal form which diffracted to 7-8 A resolution. A new crystal form was grown by using a protein variant containing mutations of two surface residues. The N-terminal sequence of the protein was also varied to reduce heterogeneity which was detected by protein mass spectrometry. A further crystallisation search using the double mutant protein and varying the RNA hairpins resulted in crystals diffracting to beyond 1.7 A. The methods and strategy described in this paper may be applicable to crystallisation of other RNA-protein complexes.

Amino Acid Sequence↗
PubMed1997

Characterization of the components and activity of Sonchus yellow net rhabdovirus polymerase.

Sonchus yellow net virus (SYNV) is the best-characterized member of a group of plant rhabdoviruses that replicate in the host cell nucleus. Using a recently developed method for partial purification of active SYNV polymerase by salt extraction of nuclei from infected plant tissue (J. D. O. Wagner et al, J. Virol. 70:468-477, 1996), we have identified the nucleocapsid (N), M2, and L proteins as polymerase complex components (based on copurification with the polymerase activity and by coimmunoprecipitation assays). Furthermore, the L protein was shown by antibody inhibition analysis to be a functional component of the polymerase. A second complex of M2 and L proteins, thought to be a precursor to the polymerase complex, was also identified. In addition, we conducted a detailed characterization of SYNV RNA synthesis in vitro. The results demonstrate that the RNAs are transcribed sequentially, beginning with the N mRNA and followed successively by the remaining five mRNAs in the order of their genome organization. Gene expression conforms to a cascade pattern, with synthesis of the 3'-proximal N mRNA occurring at the highest level, followed by consecutively lower levels of transcription from each subsequent gene. The reaction conditions favor transcription over minus-sense RNA replication, which, we posit, is inhibited near specific signal sequences located on the antigenomic template. The results support the concept that the mechanism of transcription is highly conserved among diverse rhabdoviruses and are compatible with a unified model for the regulation of genomic and antigenomic RNA synthesis.

Antibodies, Viral↗
PubMed1999

Mitochondrial DNA acts as potential promoter of the baculovirus RNA polymerase.

We have examined whether mitochondrial DNA could act as target of the RNA polymerase encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus, because the baculovirus late promoters and the control region of host mitochondrial DNA show a high degree of sequence similarity. In vitro transcription using mitochondrial DNA from Spodoptera frugiperda cells and nuclear extracts prepared from baculovirus infected cells demonstrates that mitochondrial DNA is recognized by the viral RNA polymerase. Transcriptional initiation occurs at TAAG sequences, although not all of the six TAAG motifs present in the mitochondrial DNA fragment are recognized. The TAAG motif in the control region served as weak transcriptional start site, but some of the TAAG motifs in the coding sequences of the adjacent tRNA and rRNA genes are recognized efficiently. The sequences flanking the TAAG motifs used as transcriptional start sites have a lower helix stability than the flanking sequences of the nonfunctional TAAG motifs. These results support the view that helix stability rather than sequence specificity is an important factor for recognition of TAAG motifs by the viral RNA polymerase.

Animals↗
PubMed1988

RNA polymerases B and C are more closely related to each other than to RNA polymerase A.

Amino acid sequence comparison of the largest subunit of the three forms of yeast nuclear RNA polymerase disclosed six major conserved regions that are partly retained in the cognate subunits from bacteria, viral, and insect enzymes (Mémet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. (1988) J. Biol. Chem. 263, 2830-2839). Within these conserved domains, the high sequence similarity of B220 and C160 subunits (52% identity) sets them apart from yeast enzyme A subunit A190. Parsimony analysis at the gene and protein levels suggests the existence of a transient ancestor to eukaryotic RNA polymerases B and C. These results are discussed in the light of the recent finding of class C genes containing RNA polymerase B promoter elements.

Amino Acid Sequence↗
PubMed1997

Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays.

Methods for identifying the genotype of hepatitis C virus (HCV) in clinical specimens are frequently based upon the direct characterisation of viral RNA sequences by polymerase chain reaction (PCR) amplification, or by serologically based methods, in which the infecting genotype is inferred from the pattern of antibody reactivity to type-specific peptides or recombinant proteins used as antigens in an Enzyme Linked Immunosorbent Assay (ELISA). Although genotyping by direct, PCR-based methods show generally highly concordant results with the genotype inferred from serological typing assays (> 95% agreement), there exist a small number of samples that produce discrepant results. To investigate the underlying reasons for the discrepancies, we obtained eleven samples from haemophiliacs and four samples from patients with chronic hepatitis C that produced discordant results between a PCR based assay (InnoLipa I and II) and a serotyping assay (Murex HC02). Nucleotide sequences in the 5'noncoding region (5'NCR), core, and NS4 region were used to identify the genotype of the circulating virus and to identify amino acid changes in NS4 that might alter antigenicity. In 14 samples, sequence analysis of all three regions was concordant with the results of the InnoLipa assay. There were few if any amino acid substitutions in NS4 that might have accounted for the discrepant serotyping results, which were found predominantly in samples from individuals with a history of multiple exposure to HCV. It remains unclear whether the detection of antibody in such discrepant samples corresponds to previous expression of a different genotype than detected by PCR, or whether the virus population in plasma is more restricted in genotype diversity than the population in the liver or at other sites of viral replication.

Amino Acid Sequence↗
PubMed1996

Sequences homologous to yeast mitochondrial and bacteriophage T3 and T7 RNA polymerases are widespread throughout the eukaryotic lineage.

Although mitochondria and chloroplasts are considered to be descendants of eubacteria-like endo- symbionts, the mitochondrial RNA polymerase of yeast is a nucleus-encoded, single-subunit enzyme homologous to bacteriophage T3 and T7 RNA polymerases, rather than a multi-component, eubacterial-type alpha 2 beta beta' enzyme, as encoded in chloroplast DNA. To broaden our knowledge of the mitochondrial transcriptional apparatus, we have used a polymerase chain reaction (PCR) approach designed to amplify an internal portion of phage T3/T7-like RNA polymerase genes. Using this strategy, we have recovered sequences homologous to yeast mitochondrial and phage T3/T7 RNA polymerases from a phylogenetically broad range of multicellular and unicellular eukaryotes. These organisms display diverse patterns of mitochondrial genome organization and expression, and include species that separated from the main eukaryotic line early in the evolution of this lineage. In certain cases, we can deduce that PCR-amplified sequences, some of which contain small introns, are localized in nuclear DNA. We infer that the T3/T7-like RNA polymerase sequences reported here are likely derived from genes encoding the mitochondrial RNA polymerase in the organisms in which they occur, suggesting a phage T3/T7-like RNA polymerase was recruited to act in transcription in the mitochondrion at an early stage in the evolution of this organelle.

Amino Acid Sequence↗
PubMed1994

Start site selection at lacUV5 promoter affected by the sequence context around the initiation sites.

The effects of single base pair substitutions at the initiation sites of lacUV5 promoter on the transcription start site selection by E. coli RNA polymerase were systematically studied. Transcription start sites were mapped by sizing the cytosine-specifically terminated transcripts produced in vitro by using a chain terminator 3'-deoxycytidine 5'-triphosphate (3'-dCTP) in transcription reactions. Transcription of a prototype lacUV5 promoter initiated with three purines (-1G, +1A and +2A; +1 representing the predominant start site) located 6-8 bp downstream from the Pribnow box. All the substitutions affected the start site selection, resulting in a change in the number of start sites (from 3 to 2 or 1) and/or a shift of the major start site (to -1 or +2). None of the variants started outside the 3-bp region and at the positions substituted by a pyrimidine. Purine-to-pyrimidine changes suppressed not only initiation at the substituted position but also, in some cases, at the other purine position. Purine-to-purine changes also shifted the major start site or suppressed the initiation at other sites. Changes at -2 and +5 also affected the start site selection. Thus, the sequence context around the initiation sites of lacUV5 promoter strongly influences the selection of initiating nucleotides by E. coli RNA polymerase.

Base Sequence↗
PubMed1994

Basepairing with 18S ribosomal RNA in internal initiation of translation.

In concert with the translation initiation factors 'trans-acting' factors function specifically during internal initiation on picornaviral mRNAs. Of these trans-acting factors, two have been identified as the La-protein and the polypyrimidine tract binding protein. Within the internal ribosomal entry site on the viral RNA, sequences are present that direct the ribosome to the initiation codon. We suggest that selection of the correct AUG initiation codon occurs through basepairing with a part of 18S ribosomal RNA.

Base Composition↗
PubMed1993

Repression of bacteriophage promoters by DNA and RNA oligonucleotides.

We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids rather than polypeptides. An in vitro model system involving repression of bacteriophage T7 RNA polymerase initiation has been employed to demonstrate that certain DNA oligonucleotides can repress transcription by site-specific triple-helix formation at two kinds of homopurine operator sequences [Maher, L. J., III, (1992) Biochemistry 31, 7587-7594]. Recognition in the purine motif is based on antiparallel oligonucleotide binding (G.G.C and T.A.T triplets). Recognition in the pyrimidine motif is based on parallel oligonucleotide binding (C+.G.C and T.A.T base triplets). Using this system, we report that the concentration-dependence of repression by DNA oligonucleotides provides triple-helix inhibition constant (Ki) estimates of approximately 2 x 10(-7) M for both purine motif and pyrimidine motif DNA complexes. RNA oligonucleotides are shown to repress promoters overlapping pyrimidine motif operators (Ki = 6 x 10(-7) M), but not purine motif operators. Although competent to hybridize to complementary single strands, RNA oligonucleotides fail to bind the purine motif operator. Partial substitution of deoxyribose residues tends to rescue repressor activity by RNA oligonucleotides in the purine motif. These results suggest prospects for, and constraints on, natural and artificial RNA-based repressors.

Bacteriophage T7↗
PubMed1996

Sequence-specific binding of antitumour bisquaternary ammonium heterocycles to DNA and inhibition of polymerase activity in vitro.

Ten bisquaternary ammonium heterocycles (BQA) active against experimental tumours were investigated for possible sequence-selective binding to DNA. Footprinting analyses indicated that several bound preferentially to dAdT runs consisting of at least four base pairs. Shortening of one or two spacer groups between the aromatic rings of the ligands (by replacement of CONH with NH) emerged as a prerequisite for sequence-specific binding. Other relevant factors concerned the overall shape of the ligands and the relative position of their positive charges. Footprinting plots evaluated for the BQA compound SN 6132 on the 167mer EcoRI-RsaI restriction fragment from plasmid pBR322 yielded the highest individual binding constant for the symmetrical base sequence AATTTAA, with approximate K(A) = 2.0 x 10(6)/M. Polymerase-catalysed syntheses of DNA and RNA in vitro were inhibited by all BQA derivatives, but the inhibition was much more pronounced with the sequence-specific binders SN 6999 and SN 6132 than with the non-specific ligand SN 6113.

Antineoplastic Agents↗