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PubMed1982

Viroid RNA is accepted as a template for in vitro transcription by DNA-dependent DNA polymerase I and RNA polymerase from Escherichia coli.

The RNA genome of potato spindle tuber viroid (PSTV) is transcribed in vitro into complementary DNA and RNA by DNA-dependent DNA polymerase I and RNA polymerase, respectively, from Escherichia coli. In vitro synthesis of complementary RNA produces distinct transcripts larger than unit length thus reflecting the in vivo mechanism of viroid replication. The influence of varying experimental conditions on the transcription process is studied; actinomycin D is found to drastically reduce complementary RNA synthesis from the PSTV RNA template by RNA polymerase.

DNA Polymerase I↗
PubMed1998

Site-specific modification of 4.5S RNA apical domain by complementary oligodeoxynucleotides carrying an alkylating group.

Site-specific alkylation of RNA by reactive oligodeoxynucleotides provides structural information and represents the first step towards the design of RNA derivatives to be used for functional studies. Specific alkylation of 4.5S RNA at G53, the first base of the apical tetraloop, was achieved by incubation with oligodeoxynucleotide ON2, complementary to nucleotides 38-53, which carries a p-(N-2-chloroethyl-N-methylamino)benzylamidophosphate group at the 5' end. Alkylation efficiency was increased by a factor of 6, without alteration of specificity, in the presence of a helper oligodeoxynucleotide, ON1, complementary to nucleotides 58-71 of the opposite strand of the RNA helix. A second reactive oligodeoxynucleotide, ON1-3'-R, was obtained by attaching the alkylating group to the 3' end of ON1. ON1-3'-R was able to modify G58. In the presence of ON2 as a helper oligodeoxynucleotide, the specificity of ON1-3'-R changes and efficient alkylation of nucleotides G54, A56 and G57 of the apical region of 4.5S RNA was observed.

Alkylating Agents↗
PubMed1994

Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain.

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450AROM) is an important step in the mechanism of androgen action in the brain. In adult rats, P450AROM activity (AA) is regulated by androgens in the preoptic area and medial basal hypothalamus, but is constitutive in the amygdala. This study was undertaken to determine the distribution of P450AROM messenger RNA (mRNA) and AA in adult rat brain and examine the effects of steroid treatments on their concentrations in various brain regions. AA was determined by a sensitive assay that measures the production of 3H2O during the conversion of [1 beta-3H]androstenedione to estrone. P450AROM mRNA was measured by a ribonuclease protection assay using a RNA probe complementary to the 5'-coding region of rat P450AROM mRNA. The 32P-labeled P450AROM probe protected two mRNA fragments in brain tissues that expressed AA (preoptic area, medial basal hypothalamus, amygdala, and hippocampus). The larger protected RNA fragment was 430 nucleotides (nt) long and corresponded in size to the full-length protected complementary RNA, whereas the shorter protected RNA fragment was 300 nt long. Brain tissues that did not exhibit AA contained either the smaller protected RNA fragment (cingulate and parietal cortex) or no protected RNA (cerebellum). These results suggest that the 430-nt protected RNA fragment represents mRNA that encodes the functional P450AROM enzyme. In agreement with this conclusion, we found that immature rat ovaries that were stimulated with PMSG to synthesize estrogen contained only the 430-nt protected fragment. The levels of the 430-nt protected RNA fragment differed significantly between brain regions (amygdala > > preoptic area > medial basal hypothalamus > or = hippocampus) and were significantly correlated with AA (r = 0.994; P < 0.001). After castration, the concentrations of P450AROM mRNA and AA decreased significantly in the preoptic area and medial basal hypothalamus (P < 0.05), but not in the amygdala. Treatments with testosterone or dihydrotestosterone maintained P450AROM mRNA and AA at levels approximating those found in intact males. Although 17 beta-estradiol treatment increased AA in the preoptic area, it did not affect the P450AROM mRNA content. These results suggest that the increase in AA observed after exposure to androgens results from regulation of the transcription and/or stability of P450AROM mRNA. In contrast, estradiol appears to exert an effect on AA at the posttranscriptional level.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗
PubMed1987

Site-directed cleavage of RNA.

Using complementary chimeric oligonucleotides containing deoxyribonucleotides and 2'-O-methylribonucleotides (1), enzymatically synthesized RNA (90 mer) were cleaved at a single site with Escherichia coli RNaseH, either at a hairpin loop or at a stem region. Especially, site-specific cleavage occurred in even the target region being enclosed within a stable, base-paired stem. The method is proved to be generally applicable to RNA containing secondary structures.

Base Sequence↗
PubMed1996

Non-viral sequences at the 5' termini of mRNAs derived from virus-sense and virus-complementary sequences of the ambisense RNA segments of rice stripe tenuivirus.

The three small segments of the four RNAs of the rice stripe tenuivirus (RSV) genome have an ambisense coding strategy. The mRNA transcripts corresponding to open reading frames for the non-structural protein (NS4) and nucleocapsid protein (N), which are encoded on virus-sense (v) RNA 4 and virus-complementary sense (vc) RNA 3, respectively, were recovered from polysomes of RSV-infected wheat leaves, and their 5' termini were analysed. The mRNAs derived from both v and vc sequences contained from 10 to 23 non-viral bases at their 5' termini. Results of nucleotide sequence similarity analyses indicated that these non-viral heterogeneous sequences may be derived from host cellular mRNAs. Taken together, these results suggest that the viral mRNA transcription of either v or vc sequences of ambisense segments of RSV is primed by non-viral oligonucleotides in vivo.

Base Sequence↗
PubMed1989

Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA.

Transgenic tobacco plants that express RNA sequences complementary to the tobacco mosaic virus (TMV) coat protein (CP) coding sequence with or without the tRNA-like structure at the 3' end of the TMV RNA were produced. Progeny of self-pollinated plants were challenged with TMV to determine their resistance to infection. Plants that expressed RNA sequences complementary to the CP coding region and the 3' untranslated region, including the tRNA-like sequences, were protected from infection by TMV at low levels of inoculum. However, plants that expressed RNA complementary to the CP coding sequence alone were not protected from infection. These results indicate that sequences complementary to the terminal 117 nucleotides of TMV, which include a putative replicase binding site, are responsible for the protection. However, the level of protection in these plants was considerably less than in transgenic plants that expressed the TMV CP gene and accumulated CP. Since the mechanisms of protection in the two systems are different, it may be possible to increase protection by introducing both sequences into transgenic plants.

Molecular Weight↗
PubMed1985

Nucleotide sequence of cucumber mosaic virus RNA. 1. Presence of a sequence complementary to part of the viral satellite RNA and homologies with other viral RNAs.

The nucleotide sequence of the 3389 residues of RNA 1 (Mr 1.15 X 10(6) of the Q strain of cucumber mosaic virus (CMV) was determined, completing the primary structure of the CMV genome (8617 nucleotides). CMV RNA 1 was sequenced by the dideoxy-chain-termination method using M13 clones carrying RNA 1 sequences as well as synthetic oligonucleotide primers on RNA 1 as a template. At the 5' end of the RNA there are 97 noncoding residues between the cap structure and the first AUG (98-100), which is the start of a single long open-reading frame. This reading frame encodes a translation product of 991 amino acid residues (Mr 110791) and stops 319 nucleotide residues from the 3' end of RNA 1. In addition to the conserved 3' region present in all CMV RNAs (307 residues in RNA 1), RNAs 1 and 2 have highly homologous 5' leader sequences, a 12-nucleotide segment of which is also conserved in the corresponding RNAs of brome mosaic virus (BMV). CMV satellite RNA can form stable base pairs with a region of CMV RNAs 1 and 2 including this 12-nucleotide sequence, implying a regulatory function. This conserved sequence is part of a hairpin structure in RNAs 1 and 2 of CMV and BMV and in CMV satellite RNA. The entire translation products of RNA 1 of CMV and BMV could be aligned with significant homology. Less prominent homologies were found with alfalfa mosaic virus RNA 1 translation product and with tobacco mosaic virus Mr-126000 protein.

Amino Acid Sequence↗
PubMed1980

Expression of the genome of defective interfering pseudorabies virions in the presence or absence of helper functions provided by standard virus.

The synthesis of virus RNA and proteins in cells infected with two populations of defective virions [Pr(1)53 and Pr(2)53] which vary in the overall composition of their DNA, but which share some structural and biological characteristics, have been examined. The experiments were done under two sets of conditions: (1) at high multiplicity of infection. In this case, practically all the cells in the cultures were co-infected with defective and infectious virions; (2) at low multiplicity of infection. In this case, 75% of the cells in the cultures were infected with defective virions only and 25% were co-infected with defective and infectious virions. The relative abundance of RNA classes complementary to different regions of the virus genomes that were synthesized under various conditions of infection were determined by the Southern (1975) technique; the synthesis of virus proteins was determined by polyacrylamide gel electrophoresis (PAGE). In cells co-infected with standard and defective virions, RNA complementary to the regions that are reiterated in the defective genomes is present in larger amounts than in cells infected with standard virions alone, indicating that the genomes of the defective virions are transcribed. Furthermore, the transcriptional controls that operate normally in the infected cells also operate in cells co-infected with standard and defective virions. The over-abundant accumulation of transcripts of some regions of the virus genome in cells co-infected with defective virions is not necessarily accompanied by an overproduction of some virus proteins. No difference in the PAGE profiles of the proteins synthesized was detected in cells co-infected with PR(1)53 and standard virions. However, cells co-infected with standard and Pr(2)53 overproduced three polypeptides. Transcription of the virus genome is detectable in cells infected with Pr(2)53 alone but not in cells infected with Pr(2)53 alone. Virus protein synthesis is also detectable under these conditions in Pr(2)-, but not in Pr(1)-infected cells. Thus, despite the similarities in the biological characteristics of the two populations of defective virions described previously, similarities with respect to the expression of their genomes were not found.

Defective Viruses↗
PubMed1996

Inhibition of rabies virus infection by an oligodeoxynucleotide complementary to rabies virus genomic RNA.

To develop antirabies virus-specific agents, eight oligodeoxynucleotides (ODN) complementary to either rabies virus genomic RNA (negative polarity) or rabies virus transcripts (mRNA) were synthesized and tested for their activity to inhibit rabies virus infection in cell cultures. It was found that the ODN RH+1 complementary to rabies virus genomic RNA blocked almost completely rabies virus infection at concentrations as low as 2 microM, whereas ODN complementary to viral transcripts did poorly even at concentrations as high as 20 microM. The antigenomic ODN also has the ability to inhibit cell-to-cell spread of rabies virus, which is an indicator for protection of rabies virus infection in vivo. These results indicate that ODN complementary to rabies virus genomic RNA have strong ability to inhibit rabies virus infection in cell culture and may have the potential to be used for therapy in clinical rabies.

Animals↗
PubMed2002

Watson-Crick base-pairing properties of tricyclo-DNA.

Tricyclo-DNA belongs to the family of conformationally restricted oligodeoxynucleotide analogues. It differs structurally from DNA by an additional ethylene bridge between the centers C(3') and C(5') of the nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. The synthesis of the hitherto unknown tricyclodeoxynucleosides containing the bases cytosine and guanine and of the corresponding phosphoramidite building blocks is described, as well as a structural description of a representative of an alpha- and a beta-tricyclodeoxynucleoside by X-ray analysis. Tricyclodeoxynucleoside building blocks of all four bases were used for the synthesis of fully modified mixed-base oligonucleotides. Their Watson-Crick pairing properties with complementary DNA, RNA, and with itself were investigated by UV melting curves, CD spectroscopy, and molecular modeling. Tricyclo-DNA was found to be a very stable Watson-Crick base-pairing system. A UV melting curve analysis of the decamers tcd(pcgtgacagtt) and tcd(paactgtcacg) showed increased thermal stabilities of up to DeltaT(m)/mod. = +1.2 degrees C with complementary DNA and +2.4 degrees C with complementary RNA. With itself, tricyclo-DNA showed an increase in stability of +3.1 degrees C/base pair relative to DNA. Investigations into the thermodynamic properties of these decamers revealed an entropic stabilization and an enthalpic destabilization for the tricyclo-DNA/DNA duplexes. CD spectroscopic structural investigations indicated that tricyclo-DNA containing duplexes preferrably exist in an A-conformation, a fact which is in agreement with results from molecular modeling.

Base Pair Mismatch↗
PubMed1999

Length dependence of RNA-RNA annealing.

The association of complementary nucleic acids can be described by a second order rate constant k. For extended molecules, including complex nucleic acids, values of k were shown to be proportional to the square root of the chain length L of the shorter nucleic acid strand at temperatures between tm and tm-30 degrees C. For homopolymers this is true over a wider temperature range. Below temperatures of tm-30 degrees C, annealing rate constants may sharply decrease due to the formation of intramolecular structures. It seems to be reasonable to assume that the formation of intramolecular structures of nucleic acids reduces the density of nucleation sites for annealing and, thereby, lowers the rates of association. Here, we examined the relationship between RNA chain length and the kinetics of RNA-RNA annealing at physiological ionic strength and temperature. We used a complete sequence space derived from chloramphenicol acetyltransferase (cat) sequences to average over all structures for each given length. For groups of progressively longer antisense RNA species and a 800 nucleotides long complementary RNA, the observed annealing rate constants kobs were measured in vitro. The structure-averaged values for kobs of RNA-RNA annealing were not related to the square root of the chain length. Instead, they were found to be proportional to 10(alphaL) (alpha=0.0017). Here, a theoretical model is suggested in which the observed length dependence is mainly influenced by ionic interactions between complementary RNA strands. The observed length dependence has substantial implications for the biological behavior of long-chain complementary RNA including the design of antisense RNA. The efficacy of antisense RNA in living cells is known to be related to annealing kinetics in vitro. Thus, on a statistical basis and independent of individual structures, long-chain rather than short-chain antisense RNA should lead to stronger inhibition.

Base Pairing↗
PubMed1995

Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides.

Antisense gene inhibition occurs when an oligonucleotide (ON) has sufficient binding affinity such that it hybridizes its reverse complementary target RNA and prevents translation either by causing inactivation of the RNA (possibly by RNase H) or by interfering with a cellular process such as stalling a ribosome. The mechanisms underlying these processes were explored. Cellular antisense inhibition was evaluated in a microinjection assay using ON modifications which precluded or allowed in vitro RNase H cleavage of ON/RNA hybrids. RNase H-independent inhibition of protein synthesis could be achieved by targeting either the 5'-untranslated region or the 5'-splice junction of SV40 large T antigen using 2'-O-allyl phosphodiester ONs which contained C-5 propynylpyrimidines (C-5 propyne). Inhibition at both sites was 20-fold less active than inhibition using RNase H-competent C-5 propyne 2'-deoxy phosphorothioate ONs. In vitro analysis of association and dissociation of the two classes of ONs with complementary RNA showed that the C-5 propyne 2'-O-allyl phosphodiester ON bound to RNA as well as the C-5 propyne 2'-deoxy phosphorothioate ON. In vitro translation assays suggested that the two classes of ONs should yield equivalent antisense effects in the absence of RNase H. Next, ON/T antigen RNA hybrids were injected into the nuclei and cytoplasm of cells. Injection of C-5 propyne 2'-O-allyl phosphodiester ON/RNA hybrids resulted in expression of T antigen, implying that the ONs dissociated from the RNA in cells which likely accounted for their low potency. In contrast, when C-5 propyne 2'-deoxy phosphorothioate ON/T antigen RNA complexes were injected into the nucleus, the duplexes were stable enough to completely block T antigen translation, presumably by RNA inactivation. Thus, a dramatic finding is that C-5 propyne 2'-deoxy phosphorothioate ONs, once hybridized to RNA, are completely effective at preventing mRNA translation. The implication is that further increases in complex stability coupled with effective RNase H cleavage will not result in enhanced potency. We predict that the development of more effective ONs will only come from modifications which increase the rate of ON/RNA complex formation within the nucleus.

Alkynes↗
PubMed2003

Structural and functional analysis of pt38, a 2.9kb plasmid of Streptococcus thermophilus yogurt strain.

The cryptic plasmid pt38 (2911 bp) of Streptococcus thermophilus ST2783, a strain isolated from Bulgarian yogurt, was subcloned and sequenced. Five ORFs (ORF1 to ORF5) were identified, although putative transcription initiation and termination signals, and Shine-Dalgarno sequence could only be localized for three of them (ORF1, ORF2, and ORF5). ORF2 would specify a 142-amino acid protein sharing a high degree of homology with plasmid-born low-molecular-weight heat stress proteins described in a variety of S. thermophilus strains. On the other hand, ORF1 would encode a 311-residue protein, which was found to be almost identical to the putative Rep proteins of previously sequenced S. thermophilus rolling circle-replicating plasmids. Intracellular single-stranded pt38 DNA was detected, showing that, in fact, the plasmid replicates via a rolling circle mechanism. A putative double-strand origin with significant homology to that of pC194, and a ssoA-type single-strand origin were also identified on the nucleotide sequence of pt38. A DNA region that can be transcribed into a small RNA (ctRNA) complementary to the leader segment of the rep (ORF1) mRNA is proposed to be involved in the control of plasmid replication. In vitro synthesis of this ctRNA was observed, and this constitutes the first report on the existence of such antisense RNAs, likely acting as regulatory elements, in S. thermophilus plasmids.

Base Sequence↗
PubMed1981

Immunochemical detection of ds-RNA in healthy and virus-infected plants and specific detection of viral ds-RNA by hybridization to labelled complementary DNA.

Double-stranded (ds) RNA was detected in nucleic acid preparations from virus-free leaf tissues of several plant species by radial double-diffusion tests with antiserum raised against polyinosinic : polycytidylic acid [poly(I) : poly(C)]. No significant differences in the concentrations of ds-RNA were detected by such tests in virus-free French bean leaves and those infected with tobacco mosaic virus or tobacco ringspot virus. However, virus-specific ds-RNAs as well as genomic RNAs of both viruses were readily detected and estimated quantitatively by hybridization to 3H-labelled complementary DNA probes. It is concluded that it is not feasible to screen plant material infected with single-stranded RNA viruses by immunochemical tests for virus-associated ds-RNAs.

Fabaceae↗
PubMed1995

In situ hybridization histochemistry of c-erbA alpha 2 mRNA in the hypothalamus and its surrounding structures in the adult male rat.

In order to detect the localization of c-erbA alpha 2 mRNA in the hypothalamus and its surrounding structures, in situ hybridization histochemistry was performed in the adult male rat. Sections through the hypothalamus were hybridized with a [3H]-labeled RNA probe complementary to c-erbA alpha 2 mRNA and then coated with photographic emulsion. Autoradiograms were then developed and localization of the hybridization signal was detected under a light microscope. Hybridization signal was detected throughout the hypothalamic and extrahypothalamic structures such as the paraventricular hypothalamic nucleus (PVN), ventromedial nucleus (VMN), arcuate nucleus (Arc), amygdala (Am), habenula (Hb), paraventricular thalamic nucleus (PVT) and hippocampus (Hipp). No hybridization signal was detected in the section which was hybridized with the sense probe. These results not only confirm previous findings in the PVN, Hb, PVT and Hipp, but also extend the finding to the VMN, Arc and Am, in which cells expressing c-erbA alpha 2 primary transcript were concentrated. These new findings may provide an important aid to the understanding of the roles of thyroid hormone in the function of the central nervous system.

Animals↗
PubMed1984

Use of complementary DNA oligomers to probe trp leader transcript secondary structures involved in transcription pausing and termination.

DNA oligomers were synthesized that are perfectly complementary to different segments of the tryptophan (trp) operon leader transcript. These 15 nucleotide long oligomers were used as probes of the involvement of transcript secondary structures in two processes: transcription pausing at the pause site located near base pair 90 in the leader region, and transcription termination at the attenuator. The 15-mers were complementary to the four segments of the trp leader transcript which have been shown to form the alternative secondary structures that are believed to be responsible for pausing, termination, and antitermination. Oligomers complementary to RNA segments 1 and 3 relieved termination while the 15-mer complementary to RNA segment 1 relieved pausing. 15-mers complementary to segment 2 had no effect on pausing and the oligomer complementary to segment 4 had virtually no effect on termination.

Base Sequence↗
PubMed1981

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.

Animals↗
PubMed1991

Kainic acid-induced seizures stimulate increased expression of nerve growth factor mRNA in rat hippocampus.

The influence of kainic acid (KA)-induced limbic seizure activity on the expression of mRNA for nerve growth factor (NGF) in adult rat brain was studied using in situ hybridization and S1 nuclease protection techniques with RNA probes complementary to murine and rat NGF mRNA. Within hippocampus, intracerebroventricular injection of 0.5 microgram KA caused a dramatic bilateral increase in hybridization of the 35S-labeled cRNA within stratum granulosum. This increase was first evident 1 h post-KA, appeared maximal at approximately 20-fold control levels at 2-3 h post-injection, and declined to control levels by 48 h post-injection. During the period of maximal hybridization, all but the deepest cells within stratum granulosum appeared to be autoradiographically labeled. Hybridization of the NGF cRNA probe was also increased within superficial layers of piriform and entorhinal cortex and, to much lesser extent, within scattered neurons of layers II and III of neocortex in KA-treated rats. In olfactory cortical areas, hybridization was maximally elevated 15.5-24.5 h after KA injection. In contrast to these effects, KA treatment did not consistently influence the density of hybridization, or number of neurons labeled, within the dentate gyrus hilus or the hippocampus proper (CA1-CA3). In agreement with the in situ hybridization results, S1 nuclease protection assay detected KA-induced increases in hybridization within pooled dentate gyrus/CA1 samples, but not hippocampal CA3 samples. These data support the conclusion that seizure activity stimulates a transient increase in NGF expression by select populations of forebrain neurons and indicates that experimental seizure paradigms might be further exploited for analyses of the mechanisms of NGF regulation and processing in the adult brain.

Animals↗