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At least 811 records · Page 45Linked to original sources
PubMed1999

Identification of new sigma K-dependent promoters using an in vitro transcription system derived from Bacillus subtilis.

In Bacillus subtilis, the genes that depend on sigma K-RNA polymerase for their transcription are expressed in the mother cell compartment at later stages of sporulation. More than a dozen genes belonging to the sigma K regulon have been identified. Here I describe the identification of two additional promoters under the control of sigma K-RNA polymerase. Using a set of histidine-tagged RNA polymerases prepared from cells harvested at various times during the course of growth and sporulation (Fujita, M., Sadaie, Y., 1998. Gene 221, 185-190), transcription initiated from putative promoter sequences on a number of DNA fragments, as inferred from genome sequencing, was examined in vitro. One of these showed sigma K-dependent transcription. For further characterization of transcription initiated from this site, in vitro transcription analysis was performed using RNA polymerase holoenzyme reconstituted from purified sigma K and core RNA polymerase. Two sigma K-dependent promoters, yfhP P1 and yfhP P2, separated by a distance of about 15 bp, were thereby identified. These promoters are located immediately upstream of the yfhP gene that encodes a protein of unknown function consisting of 327 amino acids residues. The promoter strength, the rate of open complex formation and the RNA polymerase binding affinity were examined for these two promoters in comparison with other known sigma K-dependent promoters, gerE and cotD. The promoter strength displayed was in the order of gerE > cotD > yfhP P2 > yfhP P1.

Bacillus subtilis↗
PubMed1994

Transcription of the principal sigma-factor genes, rpoD and rpoS, in Pseudomonas aeruginosa is controlled according to the growth phase.

The rpoS gene encodes the second principal sigma factor of RNA polymerase in stationary-phase cells in Escherichia coli. We examined the transcription of Pseudomonas aeruginosa rpoS as to the growth of cells. The results of quantitative S1 nuclease mapping of rpoS and rpoD, encoding the principal sigma factor, indicated that the transcription of rpoS is induced in stationary-phase cells, whereas that of rpoD is induced in exponential-phase cells. By high-resolution S1 nuclease mapping, the 5'- and 3'-ends of rpoS mRNA were determined. The results indicated that rpoS is transcribed as a monocistronic mRNA. The sequence preceding the 5' end of rpoS mRNA showed poor homology to the consensus sequences of the previously known promoters. P. aeruginosa rpoS was not transcribed in E. coli. By in vitro transcription assaying, P. aeruginosa rpoS was shown to be transcribed by the RNA polymerase fraction containing the principal sigma (sigma 70)-RNA polymerase of P. aeruginosa.

Amino Acid Sequence↗
PubMed2002

Mechanism of specific recognition of the aidB promoter by sigma(S)-RNA polymerase.

Transcription of the Escherichia coli aidB gene is controlled by an Esigma(S)-dependent promoter (PaidB) and is poorly transcribed by the Esigma(70) form of RNA polymerase in the absence of additional factors. In this report, we investigate the interaction between PaidB and either the Esigma(70) or the Esigma(S) forms of RNA polymerase in vitro. We show that although Esigma(70) can bind the aidB promoter, its interaction with the promoter results in the formation of an open complex inefficient in transcription initiation and sensitive to heparin challenge. Deletion of the C residue at position -13 of PaidB (Delta-13C) slightly impaired transcription initiation by Esigma(S), consistent with the role of -13C as a specific feature of Esigma(S)-dependent promoters. However, Esigma(S) could still bind and initiate transcription from the Delta-13C mutant aidB promoter more efficiently than Esigma(70), suggesting that sequence elements other than the -13C play an important role in specific promoter recognition by Esigma(S).

Bacterial Proteins↗
PubMed1994

Determinants of serotype specificity in transcription of vesicular stomatitis virus synthetic nucleocapsids.

Transcription of the nucleocapsid template of vesicular stomatitis virus (VSV) is serotype specific, since the viral RNA polymerase of the VSV Indiana (Ind) serotype transcribes the Ind nucleocapsid but not the nucleocapsid of the VSV New Jersey (NJ) serotype and, similarly, the NJ RNA polymerase transcribes only the NJ nucleocapsid. We have prepared synthetic nucleocapsid templates from various combinations of purified leader gene RNA and N protein of these two VSV serotypes for analysis of serotype specificity in vitro. In agreement with previous observations, in vitro transcription of synthetic homologous nucleocapsids with the 3' terminal leader RNA gene of either the (+) or (-) strand sense and the N protein of the same serotype is also serotype specific. We show with chimeric nucleocapsids, where the RNA and N protein are of different serotypes, that both components of the template are important for specificity, although the specifics depend on the serotype from which the RNA polymerase is derived. The Ind RNA polymerase will transcribe only the homologous nucleocapsids where both the RNA and N protein are of the Ind serotype, and not the chimeric nucleocapsids. In contrast, the NJ RNA polymerase is active not only on the homologous synthetic nucleocapsid, but also gives significant levels of transcription as long as one of the nucleocapsid components (N protein or RNA) is of the NJ serotype. The divergent RNA sequence of the distal portion of the leader gene (nt22 to 50/51) seems to be a major determinant for specificity, since transcription of synthetic nucleocapsids containing just the conserved proximal 1-22 nucleotides is significantly less serotype-restricted. Restriction appears to occur at the level of elongation of the product rather than initiation of RNA synthesis, since synthesis of template-length RNA, but not reiterated small initiation products, is inhibited. In addition, the serotype of the P protein subunit of the RNA polymerase also contributes to the serotype specificity of transcription, since the NJ P protein can bind equally to NJ and Ind nucleocapsids, while Ind P protein binds preferentially to Ind nucleocapsids.

Animals↗
PubMed1994

Stalling of Escherichia coli RNA polymerase in the +6 to +12 region in vivo is associated with tight binding to consensus promoter elements.

Three synthetic promoters, PS1, PS2 and PS3, which differ in their core promoter elements, were studied in vivo and in vitro. Whereas an increased homology score correlates with higher rates of RNA polymerase binding, it does not correlate with activity in vivo. Permanganate probing in vivo reveals that PS1, which exhibits the lowest homology score, is rate-limited during the early phase of promoter-RNA polymerase interactions. By contrast, PS2 and PS3, with higher homology scores, are limited at a late step involving an open DNA region spanning from +6 to +12, indicating a stalling of RNA polymerase. These complexes disappear upon treatment of cells with rifampicin and are replaced by open complexes covering the start site. Because initiated complexes are selectively insensitive to rifampicin action, this confirms that RNA polymerase stalled at +6 to +12 has initiated RNA synthesis. Kinetic studies indicate that the enzyme is released slowly from this position and that this slow release appears to be responsible for the low promoter activity. For PS3, which exhibits the highest homology score and which binds RNA polymerase most efficiently, the release of the stalled complex is particularly slow. PS3 is found to be the weakest of the three promoters in vivo. These results support models in which promoter activity can be determined by various rate limiting steps, including those following the formation of open complexes and even the initiation of RNA synthesis.

Base Sequence↗
PubMed1997

Molecular cloning of cDNA encoding two types of pituitary gonadotropin alpha subunit from the goldfish, Carassius auratus.

Two types of cDNA encoding the alpha subunit of pituitary gonadotropin (GTH) were cloned by polymerase chain reaction (PCR) for goldfish pituitary cDNA library. The goldfish GTH-alpha subunit cDNAs, designated as alpha 1 and alpha 2, encode 117 and 118 amino acids, respectively, including a 23-amino-acid signal peptide. These two types of cDNAs showed high homology in nucleotide and amino acid sequence, but deletion of a triplet nucleotides was present in alpha 1. Deduced amino acid sequences represented differences in four residues between alpha 1 and alpha 2 subunits. Analyses of goldfish genomic DNA revealed that each individual goldfish possesses two distinct genes relevant to GTH-alpha 1 and -alpha 2 subunits. Results of reverse transcriptase-PCR analysis suggest that these two GTH-alpha subunit genes are expressed at different levels without relation to sex and gonadal maturity.

Amino Acid Sequence↗
PubMed1995

Characterisation of foscarnet-resistant strains of human immunodeficiency virus type 1.

Foscarnet is a broad-spectrum viral DNA polymerase inhibitor active in vitro and in vivo against human immunodeficiency virus type 1 (HIV-1). Strains of HIV-1 resistant to foscarnet were selected by in vitro passage in increasing concentrations of drug. Reduced susceptibility to foscarnet was evident at the levels of both HIV-1 replication and reverse transcriptase. Biologically cloned, foscarnet-resistant strains with distinct genotypes were hypersensitive to zidovudine, azidodeoxyuridine, nevirapine, and R82913 but had unchanged susceptibility to zalcitibine and didanosine. The reverse transcriptase of foscarnet-resistant strains had unique substitutions Glu89-Lys, Leu92-Ile, or Ser156-Ala, the third being associated with six polymorphic changes. Introduction of these mutations into wild-type HIV-1 by site-directed mutagenesis confirmed their role in foscarnet resistance. In the three-dimensional structure of the reverse transcriptase enzyme these amino acids are located close to the template strand of the template primer and far away from the putative pyrophosphate binding site, suggesting that the mechanism by which HIV-1 becomes resistant to foscarnet is indirect. Foscarnet resistance is thus likely to be mediated through an altered interaction of the mutant enzyme with the template strand of the template primer which distorts the geometry of the polymerase active site and thereby decreases foscarnet binding.

Amino Acid Sequence↗
PubMed1996

Persistence of Japanese encephalitis virus is associated with abnormal expression of the nonstructural protein NS1 in host cells.

Persistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (10(3) to 10(4) PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1' were produced in C2-2 cells. Both truncated NS1 and NS1' contain deletions at their N-termini; however, the analyses by RT-PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus-cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed.

Amino Acid Sequence↗
PubMed1991

Retrotransposon Gypsy and genetic instability in Drosophila (review).

The laboratory mutator strain (MS) has properties which can be characterized as genetic instability. It exhibits the high level of gypsy autonomous transposition in somatic and germ cells. This paper summarizes all the data concerning this system and gypsy itself that has been obtained in our works during the last years.

Animals↗
PubMed1990

Comparative analysis of the retroviral pol and env protein sequences reveal different evolutionary trees.

Phylogenetic trees of two different retroviral gene products were constructed by using the progressive alignment method. The reverse transcriptase domain and the amino-terminal region of the transmembrane env protein, including the fusagenic peptide domain of at least one member of each of the established retrovirus subfamilies or groups, were separately aligned. The resulting phylogenetic tree of the reverse transcriptase was compared to that of the transmembrane protein. The similarities and differences are discussed with respect to the genetic events that occurred during the evolution of retroviruses. A segment of the central part of the viral env protein sequence was found to have 22% to 37% homology with some members of the immunoglobulin superfamily.

Amino Acid Sequence↗
PubMed1986

An additional class II intron with homology to reverse transcriptase in rapidly senescing Podospora anserina.

Senescence in Podospora anserina is maternally inherited and the parameters of senescence are race specific. We have compared the restriction enzyme fragment maps of race A, the most rapidly senescing race, with race s and have found three inserts in race A which are not present in race s mitochondrial DNA. Fragment A was mapped and found to be located downstream of the so-called alpha senDNA, a class II intron, near the 5' end of the COI gene, separated from alpha senDNA by two class I introns. DNA sequence analysis showed that fragment A is also a class II intron, but with only 10% DNA sequence homology to alpha senDNA. Like alpha senDNA, intron A contains significant amino acid homology with known reverse transcriptases. The importance of this additional class II intron in the mitochondrial genome with the relative rate of senescence in race A is discussed.

Amino Acid Sequence↗
PubMed1990

Odontoglossum ringspot virus coat protein: sequence and antigenic comparisons with other tobamoviruses.

Comparative immunochemical analysis of different tobamoviruses indicated that the previously reported coat protein sequence of Odontoglossum ringspot virus was likely erroneous. This sequence has been determined again by direct sequencing of the genomic RNA and was found to differ from the previously proposed sequence in 31 of the 157 amino acid residues. The extent of antigenic cross-reactivity between ORSV protein and other tobamovirus proteins was measured by ELISA and found to correlate satisfactorily with the degree of sequence homology.

Amino Acid Sequence↗
PubMed1993

Copia-like retrotransposable element evolution in diploid and polyploid cotton (Gossypium L.).

Copia-like retrotransposable elements were identified in allotetraploid cotton, Gossypium hirsutum, and two species representing its diploid progenitors, G. herbaceum and G. raimondii. These elements are present in high copy number in all three species. Because the two diploid genomic groups have been isolated on opposite sides of the world for 6-11 million years, horizontal transfer of elements between these species is highly unlikely. Elements were intensively sampled to generate a model of copia-like retrotransposable element evolution in systems where vertical transmission is the sole probable means of descent. Copia-like retrotransposon diversity is equally great in all three Gossypium species. Despite this high heterogeneity, analysis of 89 partial reverse transcriptase sequences resulted in the recognition of nine sharply differentiated retrotransposon lineages, each containing elements that share high sequence similarity. No evidence of horizontal transfer from other taxa was obtained. Phylogenetic analyses demonstrate that element topologies are incongruent with Gossypium phylogeny. Consideration of processes that obscure phylogenetic reconstruction of multigene families (including sampling error, variable degrees of orthology and paralogy, differential lineage age and lineage loss and/or proliferation) demonstrates that incongruence between organismal and retrotransposon trees is expected under conditions in which vertical processes are the sole means of transmission. Identification of closely related elements between species allowed rates of copia-like retrotransposon sequence evolution to be estimated as approximately 10(-9) nucleotide substitutions/site/year. These rates are consistent with the interpretation that these retrotransposons have been evolving under functional constraints for most of the time frame bracketed by the species studied. Extrapolation of these results to previous studies that sampled from more highly divergent taxa indicates that horizontal transfer need not be invoked to explain observed phylogenetic patterns.

Amino Acid Sequence↗
PubMed1998

Identification of three ABC transporter genes in Trypanosoma brucei spp.

ABC transporters are key players in the multidrug resistance of cancer cells and yeast, and they appear to be involved in the drug resistance of various pathogenic protozoa. No member of this ubiquitous protein family has yet been described in Trypanosoma brucei spp., the causative agents of African sleeping sickness and animal trypanosomiases. However, different cases of artificially induced drug resistance were shown to be linked to a reduction in net drug uptake. We used polymerase chain reaction with degenerate oligonucleotide primers corresponding to particularly conserved regions within the ATP-binding cassette to probe the genome of T. brucei spp. for the presence of ABC transporter genes. Three different sequence segments encoding ATP-binding cassettes were identified, which, upon Southern blotting, appeared to belong to distinct genes designated Tbabc1, Tbabc2, and Tbabc3. They appear to be single-copy genes in both drug-susceptible and drug-resistant stocks of T. brucei spp., expressed in bloodstream forms as well as in the procyclic life stage. Whereas Tbabc3 shows moderate homology to various known ABC transporters, Tbabc1 and Tbabc2 are highly homologous to P-glycoprotein A of Leishmania tarentolae and to the multidrug resistance protein 1 of L. donovani, respectively.

ATP-Binding Cassette Transporters↗
PubMed2005

Variable regions 1 and 2 (VR1 and VR2) in JSRV gag are not responsible for the endogenous JSRV particle release defect.

Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus causing ovine pulmonary adenocarcinoma, a transmissible lung tumor of sheep. A very closely related endogenous retrovirus (enJSRV) occurs as 15 to 20 copies in the genome of all sheep, and is not known to be linked to pathogenesis. We previously localized a particle release defect of the full-length endogenous-derived expression construct pCMV2enJS56A1 to the amino-terminal region of gag that incorporates the two variable regions VR1 and VR2, which harbor the main sequence differences between endogenous and exogenous JSRV in this part of gag. Here, we tested the hypothesis that either or both of these variable regions are responsible for the observed particle release defect in enJS56A1. We found that the PPPPPPPS motif of the exogenous VR1 is neither necessary nor sufficient for particle release. Furthermore, the precise substitution of VR1 and VR2 in the exogenous JSRV expression plasmid pCMV2JS21, using their enJS56A1-derived counterparts, did not abrogate the ability of the resulting constructs to release particles. The particle release defect of enJS56A1 is therefore not determined exclusively by either VR1 or VR2. These results point to a small number of amino acids lying outside of VR1 and VR2 that may be responsible for the particle defect of enJS56A1 Gag.

Amino Acid Sequence↗
PubMed1994

Selectivity of DNA polymerases toward alpha and beta nucleotide substrates of D and L series.

The substrate properties of four carbocyclic D and L nucleoside 5'-triphosphate analogs toward HIV and AMV reverse transcriptases and terminal deoxynucleotidyl transferase were evaluated. The compounds of the D-beta and L-beta series were found to be terminating substrates for these enzymes, while the derivatives of the D-alpha and L-alpha series were recognized only by terminal deoxynucleotidyl transferase, suggesting that for the template-independent enzyme the mutual orientation of the two fragments is of no significance. A hypothesis for binding of nucleotides to the DNA polymerase active center was proposed.

Adenosine Triphosphate↗
PubMed1988

Retrotransposon-like sequences are expressed in Y chromosomal lampbrush loops of Drosophila hydei.

The DNA sequence family micropia consists of repeated DNA sequences that occur dispersed in the genome of Drosophila hydei. Members of this DNA sequence family were recovered from two recombinant DNA clone banks obtained by microdissection of the two Y chromosomal lampbrush loop threads and pseudonucleolus from primary spermatocyte nuclei. Nucleotide sequence analysis of two of the recombinant DNA clones revealed homology to the DNA region coding for a reverse transcriptase-like protein in retroviruses and retrotransposons. Homologous tissue-specific transcripts of a size of 1.2 x 10(3) base-pairs were found in testes. Transcript in-situ hybridization shows that at least parts of these transcripts are synthesized in these Y chromosomal lampbrush loops, which were originally used for microdissection. Also the cytoplasm of primary spermatocytes contains homologous RNA species. These observations are discussed in the context of lampbrush loop function and evolution.

Amino Acid Sequence↗