Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence spectroscopy

Antisense strategy has high potential for curing diseases and studying gene functions by suppressing the translation step. For the strategy, it is essential to detect acceptor sites of antisense molecules on mRNA under physiological conditions. We propose a new analytical method for the detection of acceptor sites of antisense molecules with high sensitivity. 2'-O-Methyloligoribonucleotide containing 2'-O-(1-pyrenylmethyl)uridine (OMUpy) was chosen as the fluorescence probe. The fluorescence intensity due to the pyrene in single-stranded OMUpy was scarcely observed. When OMUpy was hybridized with the complementary oligoRNA, the fluorescence intensity at 375 nm was remarkably increased. It was found that the increase was derived from the localization of the pyrene by the measurements of time-resolved fluorescence spectroscopy, CD and UV absorption spectra. These results suggest that the change of the fluorescence intensity of OMUpy can be a useful index to monitor hybridization. In this study, we chose Escherichia coli. 16S-rRNA as the model RNA and chose seven regions for probing by OMUpy based on the reported secondary structure of 16S-rRNA. The fluorescence intensity of an equimolar mixture of OMUpy with 16S-rRNA varied depending on the sequence. In particular, the increment in the system of OMUpy-8, which can hybridize with region 887-896 nt of 16S-rRNA, was most significant among the systems. These results indicated that the site targeted by OMUpy-8 was exposed to regulatory molecules, and suggest that the method presented here is useful to design antisense molecules.     

Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis

Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B. bacilliformis. The activity was not apparent in culture supernatants but was reliably detected when B. bacilliformis cells were centrifuged onto erythrocytes prior to incubation. Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein. Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein. A preparation containing factors released by B. bacilliformis, including deformin, a B. bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes. However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B. bacilliformis cells. Inhibition of deformation caused by B. bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity. This study suggests hemolytic activity and deforming activity are attributable to different B. bacilliformis proteins.     

Taxonomic description of Methanococcoides euhalobius and its transfer to the Methanohalophilus genus

A sequence analysis of the 16S-rRNA of Methanococcoides euhalobius revealed that this organism was highly related to members of the genus Methanohalophilus. On the basis of sequence data, an oligonucleotide probe specific to Methanohalophilus species was designed. Hybridization studies with this probe confirmed close relationship of Methanococcoides euhalobius to Methanohalophilus species. Therefore, we propose that Methanococcoides euhalobius should be transferred to the genus Methanohalophilus as Methanohalophilus euhalobius.     

The nucleotide sequence at the 3'-end of Neurospora crassa 18S-rRNA and studies on the interaction with 5S-rRNA.

The sequence of more than 100 nucleotides at the 3'-end of Neurospora crassa 18S-rRNA was determined by chemical sequencing techniques. Extensive homologies with 18S-rRNA from other eukaryotes were found. Inspection of the nucleotide sequence at the 3'-end of N. crassa 5S-rRNA revealed the presence of sequences complementary to a region near the 3'-terminus of 18S-rRNA. Under the appropriate conditions a complex was formed between 18S-rRNA and 5S-rRNA (Tm 53 degrees C). Interaction was detected between 5S-rRNA and a specific 3'-terminal fragment from 18S-rRNA and between 18S-rRNA and a specific 3'-terminal fragment from 5S-rRNA. These findings are consistent with the idea that intermolecular base-pairing between nucleotides at the 3'-ends of 18S-rRNA and 5S-rRNA may be functionally important within the ribosome. Further investigation revealed that this intermolecular base-pairing is not essential for ribosome stability.     

Construction of the physical maps of Mycoplasma hyopneumoniae and Mycoplasma flocculare and the location of rRNA genes on these maps.

The genomes of Mycoplasma flocculare and Mycoplasma hyopneumoniae, two mycoplasmas of the porcine respiratory system, were studied. Based upon antigenic cross-reactivity and DNA-DNA hybridization, these species have given indication of a close genetic relationship. By using field-inversion gel electrophoresis and employing the restriction digest fragments obtained from the gels as the probes, physical maps of the genomes of the two species were constructed. Mycoplasma hyopneumoniae is similar to M. flocculare in having a single set of rRNA genes and the 5S-rRNA gene is separated from the 16S and 23S rRNA genes. Based upon the location of the rRNA genes on the physical maps in both species, the distance between the 5S and the 16S and 23S rRNA genes is at least 150 kbp. Thus, there is further evidence for the close relationship between these organisms.     

A phage in Bartonella bacilliformis.

Bacteriophage-like particles were found in Bartonella bacilliformis culture. The particles consisted of head (icosahedral), 40 nm in diameter, and tail, 16 nm in length.     

The nucleotide sequence at the 3''-end of Neurospora crassa 25S-rRNA and the location of a 5.8S-rRNA binding site

The sequence of 110 nucleotides adjacent to the 3'-end of Neurospora crassa 25S-rRNA has been derived by chemical sequencing methods. Sequences present between 40 and 85 nucleotides of the 3'-end were found to complement sequences at the 3'- and 5'-ends of 5.8S-rRNA. Interaction was shown to occur between 5.8S-rRNA and a specific 3'-terminal fragment of 85 nucleotides derived from 25S-rRNA. We have also demonstrated that the nucleotide sequence at the 3'-end of N. crassa 5.8S-rRNA (-UCAUUOH) is different from the published sequence (-UUUUOH) which was derived from rDNA.     

Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha-proteobacteria

Bacillary angiomatosis (BA) and chronic bartonellosis are bacterial infections of humans which result in an unusual vascular proliferative tissue response. In order to determine their phylogenetic relationships, we have determined greater than 95% of the 16S rRNA sequences for these two organisms by amplification directly from infected BA tissue and from a Bartonella bacilliformis lyophilized culture. The BA agent and B. bacilliformis are closely related alpha-proteobacteria (98.5%), although the BA agent is more closely related to Rochalimaea quintana (99.1%). Contrary to previous belief, the BA agent is distinct from, and less closely related to, the cat scratch bacillus (Afipia felis) (90.7%). We propose a novel secondary structure in a hypervariable region of the 16S rRNA which is useful for alignment of primary sequences and which may be useful for design of nucleic acid probes.     

Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain

About 300 species and varieties of Astragalus are identified in China, making the identification of the origin of a particular Astragalus species on the consumer market difficult. A molecular genetic approach was developed to identify various species of Astragalus. Although the 5S-rRNA coding sequence is conserved in higher eukaryotes, the spacer domain of the 5S-rRNA gene has great diversity among different species. The 5S-rRNA spacer domain was amplified by polymerase chain reaction (PCR) from the isolated genomic DNA, and the PCR products (approximately 300 bp) covering the 5S-rRNA spacer domain were sequenced. The nucleotide sequences of Astragalus membranaceus, A. membranaceus var. mongholicus, A. lehmannianus, A. hoantchy, and of one closely related species Hedysarum polybotrys (Hongqi), were determined. Diversity in DNA sequence and restriction enzyme mapping among various species was found in their 5S-rRNA spacer domains. This is the first report on the detection of 5S-rRNA spacer region sequence of Astragalus, and the results could be used for genetic identification of Huangqi.     

Inhibitory effect of 18S-rRNA from mouse reticulocytes on translation of exogenous and endogenous mRNA in a cell-free system of wheat embryos]

The effect of 18S-rRNA from mouse reticulocytes on the translation of exogenous and endogenous matrices in a system of wheat embryos was studied. It was found that after addition of 18S-rRNA the translation of exogenous and endogenous mRNAs is strongly inhibited. Treatment of 18S-rRNA with proteinase K prior to the addition did not remove the inhibiting action of rRNA. A RNA hydrolysis by alkali resulted in a considerable decrease of the inhibitory effect.     

Molecular cloning and analysis of a region of the Bartonella bacilliformis genome encoding NlpD,L-isoaspartyl methyltransferase and YajC homologs

The NlpD/LppB homolog of the human pathogen, Bartonella bacilliformis, is an immunogenic 43-kDa protein that is encoded by a 1206-bp open reading frame (ORF-401). The regions flanking the nlpD/lppB gene of B. bacilliformis were sequenced to determine if it is located within the rpoS operon, as it is in most bacteria. We report that the B. bacilliformis nlpD/lppB gene is located immediately downstream of pcm, a gene encoding a 25-kDa protein, L-isoaspartyl protein carboxyl methyltransferase, that is a component of the rpoS operon in other bacteria. However, the genomic organization downstream of the B. bacilliformis nlpD/lppB gene appears to be distinct. In other bacteria, the third gene in the operon is rpoS, a gene that codes for an alternative sigma factor of RNA polymerase. In B. bacilliformis, an open reading frame encoding a protein homologous to the immunodominant YajC protein is located directly downstream of the nlpD/lppB gene. We show that Bartonella henselae, a close relative of B. bacilliformis, also shares this unusual organizational feature. Thus, the genomic organization of the nlpD/lppB genes of B. bacilliformis, and B. henselae appears to be unique among all bacteria for which the sequence of this region has been reported.     

Differentiation of Bartonella species using restriction endonuclease analysis of PCR-amplified 16S rRNA genes

Abstract Differentiation of the four Bartonella species which were formerly classified as Rochalimaea using restriction endonuclease analysis of PCR-amplified citrate synthase gene fragments has previously been described. However, attempts to extend this method to include all members of Bartonella were confounded when amplification of the gene fragment from strains of B. bacilliformis each yielded two products of differing sizes. An alternative differentiation scheme for Bartonella species was developed based on restriction endonuclease analysis of their 16S rRNA genes. As the complete 16S rRNA gene sequences of all extant Bartonella species are available, the usefulness of specific endonucleases could be theoretically predetermined rather than discovered empirically. The potential usefulness of the restriction enzymes Ddel and Mnll was established using this approach, and this potential was confirmed in practice as all eight species could be distinguished from each other.     

Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies

The abundance of six tetracycline resistance genes tet(O), tet(Q), tet(W), tet(M), tet(B) and tet(L), were quantified over time in wastewater lagoons at concentrated animal feeding operations (CAFO) to assess how feedlot operation affects resistance genes in downstream surface waters. Eight lagoons at five cattle feedlots in the Midwestern United States were monitored for 6 months. Resistance and 16S-rRNA gene abundances were quantified using real-time PCR, and physicochemical lagoon conditions, tetracycline levels, and other factors (e.g. feedlot size and weather conditions) were monitored over time. Lagoons were sorted according to antibiotic use practice at each site, and designated as 'no-use', 'mixed-use' or 'high-use' for comparison. High-use lagoons had significantly higher detected resistance gene levels (tet(R); 2.8 x 10(6) copies ml(-1)) relative to no-use lagoons (5.1 x 10(3) copies ml(-1); P < 0.01) and mixed-use lagoons (7.3 x 10(5) copies ml(-1); P = 0.076). Bivariate correlation analysis on pooled data (n = 54) confirmed that tet(R) level strongly correlated with feedlot area (r = 0.67, P < 0.01) and 'total' bacterial 16S-rRNA gene level in each lagoon (r = 0.51, P < 0.01), which are both characteristic of large CAFOs. tet(M) was the most commonly detected gene, both in absolute number and normalized to 16S-rRNA gene level, although tet(O), tet(Q) and tet(W) levels were also high in the mixed and high-use lagoons. Finally, resistance gene levels were highly seasonal with abundances being 10-100 times greater in the autumn versus the summer. Results show that antibiotic use strategy strongly affects both the abundance and seasonal distribution of resistance genes in associated lagoons, which has implications on water quality and feedlot management practices.     

Development of a system for genetic manipulation of Bartonella bacilliformis.

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan(r) clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.     

A study of the influence of magnesium ions on the conformation of ribosomal ribonucleic acid and on the stability of the larger subribosomal particle of rabbit reticulocytes.

Mg2+ was shown to affect the conformation of rRNA over the range of 0.03-1.2M-KCl. The species studies were Escherichia coli S-rRNA and L-rRNA (the RNA moieties of the smaller and larger subribosomal particles respectively) and rabbits S-rRNA and L-rRNA. 2. The addition of Mg2+ to rRNA in reconstitution buffer (0.35M-KCl0.01M-Tris/HCl, pH7.2) at 20 degrees C let to an increase in bihelical secondary structure through the formation of additional (mainly A-U) base-pairs (e.g. an additional approx. 58 A-U base-pairs per molecule of E. coli S-rRNA as judged by u.v. difference spectrophotometry...     

New species of Dictyochaetopsis and Paraceratocladium from Brazil

Two new conidial fungi, Dictyochaetopsis brasiliensis and Paraceratocladium bacilliformis, both isolated from plant debris collected in Brazil, are described and illustrated. Dictyochaetopsis brasiliensis differs from the previously described species of the genus mainly by small, fusiform conidia with a very thin apical setula, absence of setae, and lateral, discrete conidiogenous cells with usually numerous sympodial proliferations. Paraceratocladium bacilliformis is characterized by small, bacilliform and aseptate conidia.     

Introns and their flanking sequences of Bombyx mori rDNA.

We obtained two different clones (16 kb and 13 kb) of B. mori rDNA with intron sequence within the 28S-rRNA coding region. The sequence surrounding the intron was found to be highly conserved as indicated in several eukaryotes (Tetrahymena, Drosophila and Xenopus). The 28S rRNA-coding sequence of 16 kb and 13 kb clone was interrupted at precisely the same sites as those where the D. melanogaster rDNA interrupted by the type I and type II intron, respectively. The intron sequences of B. mori were different from those of D. melanogaster. In 16 kb clone, the intron was flanked by 14 bp duplication of the junction sequence, which was also present once within the 28S rRNA-coding region of rDNA without intron. This 14 bp sequence was identical with those surrounding the introns of Dipteran rDNAs.     

PCR and PCR-RFLP of the 5S-rRNA-NTS region and salvinorin A analyses for the rapid and unequivocal determination of Salvia divinorum

Salvia divinorum Epling & Játiva-M. is a perennial herb belonging to the Lamiaceae family; its active ingredient, the neoclerodane diterpene salvinorin A, is a psychotropic molecule that produces hallucinations. A comparative evaluation of S. divinorum fresh and dried leaves, S. officinalis fresh leaves, and dried powdered leaves claimed to be S. divinorum was done. HPLC-MS data confirmed the presence of salvinorin A in both S. divinorun leaf extracts and the powdered leaves, whereas no salvinorin A was found in S. officinalis. The non-transcribed spacer (NTS) in the 5S-rRNA gene of all leaf samples and the dried powdered leaves was amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 500bp for S. divinorum and 300bp for S. officinalis) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences, great diversities were found in the spacer region of the two species. Specific S. divinorum primers were designed on the sequence of the 5S-rRNA gene spacer region. In addition, a PCR-restriction fragment length polymorphism (PCR-RFLP) method was applied using NdeI and TaqI restriction enzymes. An NdeI site, absent in S. officinalis, was found in S. divinorum NTS region at 428-433bp. For TaqI, multiple sites (161-164, 170-173, and 217-220bp) were found in S. officinalis, whereas a unique site was found in S. divinorum (235-238bp). The results of this work show that the combined use of analytical chemical (HPLC-MS) and molecular (DNA fingerprinting) methods lead to the precise and unequivocal identification of S. divinorum.