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PubMed1995

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Animals↗

PubMed1999

A species of small antisense RNA in posttranscriptional gene silencing in plants.

Posttranscriptional gene silencing (PTGS) is a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. Here, three types of transgene-induced PTGS and one example of virus-induced PTGS were analyzed in plants. In each case, antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Thus, the 25-nucleotide antisense RNA is likely synthesized from an RNA template and may represent the specificity determinant of PTGS.

Amino Acid Oxidoreductases↗

PubMed1994

Increased expression of tissue plasminogen activator messenger ribonucleic acid is an immediate response to parathyroid hormone in neonatal rat osteoblasts.

Osteoblasts have been reported to produce tissue-type (t) plasminogen activator (PA), which may be involved in the initiation of bone resorption via plasmin-metalloproteinase degradation of adjacent extracellular matrix. To investigate this cAMP-activated gene, we characterized the PTH regulation of tPA messenger RNA (mRNA) in neonatal rat osteoblast cultures before and after differentiation in vitro. RNA was purified from cultures at confluence or after treatment with glucocorticoid for 1 week and BGJ/ascorbic acid/beta-glycerophosphate for a second week. Northern blots of total or poly(A)+ RNA were hybridized simultaneously with an oligonucleotide or complementary RNA probe for rat tPA and an oligomeric DNA probe for cyclophilin (CYP), an abundant control gene. Differentiation was monitored by expression of rat osteocalcin mRNA and protein. Both bovine PTH1-34 and forskolin caused an increase in tPA/CYP ratio in the presence of phosphodiesterase inhibitor (IBMX) and cycloheximide (CHX). The effect was maximal (16- to 21-fold increase in tPA mRNA and 6- to 8-fold increase in tPA/CYP ratio) with 25 nM hormone for 6 h and was half-maximally stimulated by 0.75-2.5 nM PTH. The tPA response to PTH was present in first passage osteoblast cultures at confluence and after 1 to 2 weeks of glucocorticoid treatment. Exposure of the differentiated cultures of 1,25-dihydroxyvitamin D (10 nM) for 2 days markedly stimulated osteocalcin mRNA while having no effect on tPA. In Northern blots of poly(A)+ RNA from cultures not treated with CHX, IBMX and PTH (2.5 h) independently stimulated tPA mRNA with no significant effect on CYP mRNA levels. The tPA/CYP ratio increased in five consecutive experiments and the effect of IBMX and PTH were additive. These data indicate that PTH acts via cAMP to stimulate tPA expression by a mechanism that is independent of protein synthesis. The enhancement of PTH action by CHX is compatible with feedback inhibition of tPA transcription by a hormone-activated repressor (which has been proposed to occur in granulosa cells) but effects of CHX on tPA mRNA stability may also occur. Expression of tPA mRNA before and after differentiation may indicate that the enzyme has more than one function.

Animals↗

PubMed1979

Use of molecular hybridization to detect type D retrovirus markers in rhesus placentas and other tissues.

We have shown previously that approximately 20% of the Mason-Pfizer virus (MPV) genome is present as endogenous provirus in rhesus monkeys. We report here that several full-term rhesus placentas examined contain additional MPV proviral sequences in their DNA. Competitive molecular hybridization experiments demonstrated that some of these placentas also contain RNA complementary to the entire MPV 60 to 70S RNA genome. Examination of internal organs of rhesus monkeys captured in the wild also revealed the presence of additional MPV proviral sequences and expression of MPV RNA in some tissues. These results provide further evidence that MPV is being transmitted via a non-germ line mechanism in the rhesus population and now demonstrate the placenta as a good source for the identification of retrovirus transcriptional products and proviral DNA.

Animals↗

PubMed1980

Regulation of endogenous murine leukemia virus-related nuclear and cytoplasmic RNA complexity in C57BL/6J mice of increasing age.

The nucleotide sequence complexity of murine leukemia virus *MuLV)-related RNA has been measured by RNA-complementary DNA hybridization analysis in nuclear and cytoplasmic RNA isolated from liver and brain of low-leukemia-strain C57BL/6J mice of different ages. In these two tissues, an approximate 1.5- to 2-fold increase in the complexity of steadystate nuclear MuLV-related RNA sequences was observed as a function of age. Maximum complexity was observed with nuclear RNA extracts from old mice and corresponded to roughly 70 to 75% of the total MuLV genome. In contrast to the age-related increase in complexity of nuclear MuLV genome was detected in liver and brain steady-state cytoplasmic RNA, irrespective of animal age. These data suggest that control mechanisms regulating the transcription and/or stabilization of nuclear RNA transcripts of endogenous mouse MuLV-related genomes become less stringent with animal age even in low-tumor mouse strains. The data also support the existence of independent posttranscriptional mechanisms which prevent accumulation of these MuLV-related transcripts in steady-state cytoplasmic RNA and which do not seem to be as subject to the relaxation of stringency as a function of age.

Aging↗

PubMed1996

Prevention of experimental autoimmune myasthenia gravis by a monoclonal antibody to a complementary peptide for the main immunogenic region of the acetylcholine receptors.

We have previously reported that a complementary peptide (denoted RhCA 67-16), encoded by RNA complementary to that of the Torpedo acetylcholine receptor (AChR) main immunogenic region (MIR), AChR residues alpha 61-76, induces polyclonal and monoclonal Ab reactive with Ig against the AChR MIR. RhCA 67-16 vaccination also protected against the development of experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. In the present report, we found that a mAb (denoted TCM 240, IgG1 kappa) against RhCA 67-16 recognized three different idiotypic Ab (mAb 6, mAb 35, and mAb 198), which were previously reported by others to recognize the AChR MIR and to cause EAMG. Based on these results, TCM 240 was tested for prophylactic effects in EAMG. EAMG induced passively by mAb 35 was inhibited by simultaneous injection with TCM 240. The disease severity was inversely paralleled by the ratio of mAb 35 to TCM 240. EAMG induced by immunization with purified native Torpedo AChR was also inhibited by TCM 240, but not a control mAb. The inhibitory effect of TCM 240 on actively induced EAMG occurred without significantly lowering the overall AChR Ab levels, which indicates a limited repertoire of disease-causing Ab in EAMG and perhaps MG. Such findings suggest the existence of an EAMG-associated Id and also support the concept of an MIR. In a more general sense, these results demonstrate that prophylactic and perhaps diagnostic mAb for autoimmune diseases can be produced by immunization with complementary peptides for disease-associated epitopes.

Amino Acid Sequence↗

PubMed2006

Solid-phase synthesis of oligodeoxyribonucleoside boranophosphates by the boranophosphotriester method.

Oligodeoxyribonucleoside boranophosphates (BH3-ODNs), containing four kinds of nucleobases, were synthesized by the solid-phase boranophosphotriester method. The 2'-deoxyribonucleoside 3'-boranophosphate monomers having 2-cyanoethyl (CE) groups as the phosphorus protecting groups were synthesized in good yields. A new condensing reagent, 1,3-dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphate, was found to be highly effective for the condensation reaction on the solid support. We also found that 1,8-bis(N,N-dimethylamino)naphthalene could accelerate the condensation reaction without causing beta-elimination of the CE groups from the boranophosphate triesters. The internucleotidic CE groups were selectively removed by treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) under anhydrous conditions. The acetylation of the terminal 5'-hydroxy group was found to be effective to suppress the decomposition of the BH3-ODNs during the DBU treatment on the solid support. Under optimized conditions for the solid-phase synthesis and the deprotection reactions, BH3-ODNs (4mers and 12mers) containing four kinds of nucleobases were synthesized in good yields. The hybridization properties of the BH3-ODN 12mers with the complementary native DNAs and RNAs were determined by the thermal denaturing studies. In contrast to the low thermal melting (Tm) value of the duplex composed of T((PB)T)11 and native dA12 (12.8 degrees C), the duplex consisting of d(C(PB)A(PB)G(PB)T)3 and d(ACTG)3 showed a higher Tm value (44.7 degrees C) under high-salt conditions. Furthermore, d(C(PB)A(PB)G(PB)T)3 formed a more stable duplex with the complementary RNA, r(ACUG)3 with a Tm value of 50.5 degrees C. Thus, we first demonstrated that the binding affinity of BH3-ODN to a complementary DNA or RNA is dramatically increased, owing to the inclusion of the four kinds of nucleobases.

Boranes↗

PubMed1974

Attempts to detect deoxyribonucleic acid from Agrobacterium tumefaciens and bacteriophage PS8 in crown gall tumors by complementary ribonucleic acid-deoxyribonucleic acid-filter hybridization.

Labeled ribonucleic acid (RNA) complementary to Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA (cRNA) were used in a systematic study of the sensitivity of cRNA/deoxyribonucleic acid (DNA)-filter hybridization for detection of small amounts of phage or bacterial DNA immobilized on filters. A. tumefaciens cRNA of specific activity 10(6) to 2 x 10(6) counts per min per mug reacted to a significant extent when the DNA-filter contained 1% A. tumefaciens DNA in a salmon DNA background, but 0.1% A. tumefaciens DNA was not detectable. PS8 phage cRNA of the same specific activity reacted to a significant extent when the DNA-filter contained as little as 0.01% PS8 DNA in a salmon DNA background. Both kinds of cRNA were found to bind to tobacco crown gall tumor DNA-filters. Similar reaction was found with control normal callus DNA-filters but not with tobacco seedling DNA-filters. The "hybrids" formed by cRNA with normal callus and tumor DNA-filters had low thermal stability. Attempts to purify the tumor and normal callus DNA prior to immobilization on the filter resulted in elimination of this spurious binding. No evidence was found for bacterial or phage DNA in crown gall tumor DNA.

Bacteriophages↗

PubMed1987

Ontogenetic development of proenkephalin A and proenkephalin B messenger RNA in fetal pigs.

Using solution hybridization analysis and Northern blotting with complementary RNA probes labelled to high specific activity, levels of proenkephalin A and B mRNA were analyzed throughout prenatal development in the hippocampus and striatum of fetal pigs. A differential time course for the appearance of these opioid precursor mRNAs was observed: in hippocampus, both mRNAs increased linearly throughout development with proenkephalin B mRNA increasing faster than proenkephalin A mRNA. In striatum, both mRNAs behaved similarly, increasing to a maximum level around mid-gestation and declining thereafter. The differences might be attributed to differential localization of the two precursor systems in the tissues and might be of functional relevance.

Animals↗

PubMed1982

Phosphorus-31 nuclear magnetic resonance of double- and triple-helical nucleic acids. Phosphorus-31 chemical shifts as a probe of phosphorus-oxygen ester bond torsional angles.

The temperature dependence to the 31P NMR spectra of poly[d(GC)] . poly [d(GC)],d(GC)4, phenylalanine tRNA (yeast) and mixtures of poly(A) + oligo(U) is presented. The 31P NMR spectra of mixtures of complementary RNA and of the poly d(GC) self-complementary DNA provide torsional information on the phosphate ester conformation in the double, triple, and "Z" helix. The increasing downfield shift with temperature of the single-strand nucleic acids provides a measure of the change in the phosphate ester conformation in the single helix to coil conversion. A separate upfield peak (20-60% of the total phosphates) is observed at lower temperatures in the oligo(U) . poly(A) mixtures which is assigned to the double helix/triple helix. Proton NMR and UV spectra confirm the presence of the multistrand forms. The 31P chemical shift for the double helix/triple helix is 0.2-0.5 ppm upfield from the chemical shift for the single helix which in turn is 1.0 ppm upfield from the chemical shift for the random coil conformation.

Magnetic Resonance Spectroscopy↗

PubMed1977

Genome analysis of Peromyscus (Rodentia, Cricetidae) VII. Localization of satellite DNA sequences and cytoplasmic poly(A) RNA sequences of P. eremicus on metaphase chromosomes.

A satellite DNA fraction from P. eremicus, having a buoyant density of 1.705 g/ml in neutral CsCl density gradients, was isolated. In situ hybridization experiments, using 3H-RNA complementary to this DNA fraction indicated that the short (heterochromatic) arms of most of the autosomes contained this sequence. Conversely, in situ hybridization using 3H-complementary DNA (cDNA) synthesized from the cytoplasmic poly (A) RNA of P. eremicus (comprising a substantial fraction of total messenger RNA) showed that the number of silver grains in the long arms (euchromatin) was significantly higher than that in the short arms. The X chromosomes showed a distinct localization pattern of both sequences.

Animals↗

PubMed1978

Differential expression of the gene for the large subunit of ribulose bisphosphate carboxylase in maize leaf cell types.

Mesophyll cells and bundle sheath cells, the dimorphic photosynthetic cell types in the C4 plant Zea mays, differ in protein composition. In particular, the large subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carbocylase (EC 4.1.1.39) is found entirely or almost exclusively in bundle sheath cells. The DNA sequence coding for this polypeptide is contained in the chloroplast DNA of both mesophyll cells and bundle sheath cells. RNA complementary to this coding sequence has been detected in RNA from bundle sheath cells, but is almost or entirely absent from mesophyll cells. Similarly, translatable mRNA for this polypeptide has been detected in RNA from bundle sheath cells, but not from mesophyll cells. Portions of the 4200 base pair maize plastid DNA sequence Bam 9 outside the large subunit gene region appear to be transcribed in mesophyll cells. Thus differential gene expression at the mRNA level has been directly demonstrated to occur in these two cell types.

Carboxy-Lyases↗

PubMed1995

Significance of strand configuration in self-replicating RNA molecules.

The kinetic theory of replication has been extended to include dual mechanisms for conversion of self-annealed single-strand RNA to double-strand molecules, which do not replicate. An analysis of experimental results established that the replicate-template annealing reaction during transcription significantly retarded replication in vitro among three RNA variants copied by Q beta replicase. Annealing between complementary RNA strands free in solution had far less significance. The finding that an RNA variant can be replicated in a multiple hairpin configuration, but not as its single, long hairpin conformer, the correlation between stability of strand secondary structure and replicative fitness, and a lack of homology in the internal sequence of RNA variants copied by Q beta replicase support the conclusion that template competence depends on strand configuration, independent of most of the underlying base sequence. Occurrence of self-annealed strands in the Q beta replicase system was attributed to its reliance on RNA-driven strand separation, in the absence of enzyme catalysed strand unwinding. A 'configuration before sequence' path to self-replication exhibited a substantially lower combinatorial barrier than standard sequence-dependent evolution. RNA-dependent RNA synthesis in the Q beta system thus displays features of an RNA World and, interestingly, they reveal a rapid path for evolution of the first self-replicating molecule on Earth.

Base Sequence↗

PubMed1977

Genetic relationship between an influenza A and a B virus.

The base sequence homology between all eight 32P-labelled RNA segment of fowl plague virus (FPV) and the complementary RNA (cRNA) of an influenza B virus (B-mass), and between segment 8 of virus N and the cRNA of the same influenza B strain has been determined. All segments of FPV and segment 8 of virus N show a significant base sequence homology, ranging from 18 to 50% suggesting that influenza A and B viruses have a common ancestor. The conserved regions in segments 4,6 and 8 of the influenza A strains are identical to corresponding regions of the influenza B virus tested.

Base Sequence↗

PubMed1976

Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.

Animals↗

PubMed1973

Cell-free translation of paramyxovirus messenger RNA.

Polypeptides corresponding in electrophoretic mobility to virion polypeptides 1, 3, and 5 were made in a reticulocyte cell-free system to which 18S RNA from Sendai virus-infected cells was added. Immune precipitation was used to select relevant polypeptides from endogenous products. The cell-free product corresponding to virion polypeptide 3 (the nucleocapsid structure unit) was the most abundant; its tryptic peptides comigrated electrophoretically with tryptic peptides of polypeptide 3 isolated from virions. Other sedimenting classes of RNA from infected cells were tested; only the 28S fraction showed slight activity. Virion 50S RNA was inactive. These findings support the hypothesis that complementary RNA transcripts of paramyxovirion RNA are the templates for viral proteins.

Animals↗

PubMed1995

Pro-melanin concentrating hormone messenger ribonucleic acid and peptides expression in peripheral tissues of the rat.

Melanin-concentrating hormone (MCH) is a cyclic peptide which is predominantly synthetized in the hypothalamus of fish and mammalian brains. In the present paper we examined the expression of MCH mRNA and pro-MCH-derived peptides, i.e. MCH and neuropeptide-(N)-glutamic acid (E) isoleucine (I) amide (NEI), in peripheral tissues of adult rodents. By means of polymerase chain reaction (PCR) of reverse-transcribed RNA, low levels of MCH gene transcripts were detected reliably in testis, stomach, and intestine of Sprague-Dawley and Wistar rats, whereas strong expression was found in hypothalamus. Subsequent sequence analysis of the PCR products verified the authenticity of MCH mRNA found in hypothalamus and stomach. The length of MCH RNA species was measured by Northern blot and multiple MCH RNA species were detected in both rat species. Shortest polyadenylated tails were found in MCH RNAs isolated from the peripheral organs by comparison with hypothalamus MCH RNAs of Wistar rats. In order to localize MCH expression in gastrointestinal and genital tracts of Wistar rats we performed in situ hybridization with specific 33P-labeled oligoprobes joined to immunocytochemical studies with rat MCH or NEI antisera. In testis, the MCH transcripts and pro-MCH-derived peptide immunoreactivities were found at the periphery of the seminiferous tubules, suggesting expression in Sertoli cells. Studies with MCH oligoprobes and antisera directed towards MCH, NEI and alpha A-inhibin revealed similar pattern of expression in isolated Sertoli cells from Swiss mice, indicating that MCH RNA species were actually synthesized and translated in these cells. In the gastrointestinal (GI) tract, the cells expressing MCH RNA species and pro-MCH-derived peptides were predominantly expressed in the antral portion of the stomach and duodenum. Strikingly, distinct oligoprobes, recognizing antisense MCH transcript, revealed a pattern of hybridization in the GI tract similar to this observed with oligoprobes revealing the mature MCH mRNA. Furthermore, total RNA from the pyloric junction, duodenum, jejunum, ileum and hypothalamus as well appeared to contain RNA complementary to MCH mRNA suggesting therefore that antisense MCH RNA species may play a general role in regulation of MCH synthesis. Taken together, our present and previous data indicate that authentic MCH RNA species and translational products are expressed in various rodent tissues at the periphery. The cellular location suggests that MCH and associated peptides may play a role in spermatogenesis and in digestive processes.

Animals↗

PubMed1990

[Identification and characterization of the two promoters of plasmid pKYM].

Plasmid pKYM is a multicopy plasmid isolated from Shigella sonnei and multiples stably in Escherichia coli. The plasmid encodes Rep protein which is essential for its multiplication and synthesizes cop ribonucleic acid (RNA) which is a short RNA complementary to the 5' region of rep m-RNA. This RNA controls the copy number and the incompatibility of the plasmid. The previous analysis located the promoters of rep m-RNA RNA (PR) and cop RNA (PL) in the inc region. This report confirmed the presence of these promoters by analyzing the RNAs isolated from the cells carrying pKYM and those synthesized in vitro. The initiation sites of these transcriptions were also determined. Analysis of in vivo RNA suggested that the quantity of cop RNA whose size was about 90 nucleotides was larger than that of rep m-RNA and these RNAs easily formed RNA-RNA hybrid. The analysis also suggested that the synthesis of rep m-RNA was repressed by cop RNA and Rep protein itself.

Base Sequence↗