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PubMed1979

Characterization and transcription analysis of a cloned sequence derived from a major developmentally regulated mRNA of D. discoideum.

The plasmid pDd 812 contains a portion of a poly(A)+ RNA sequence isolated from developing cells of the cellular slime mold Dictyostelium discoideum (Williams and Lloyd, 1979). The poly(A)+ RNA complementary to this plasmid shows an increase in concentration during the first 4 hr of development followed by a decrease in concentration during the following 4 hr. This RNA is very abundant after 3-4 hr of development, constituting at least 2% of the poly(A)+ RNA population. In this study, we demonstrate that this poly(A)+ RNA is an mRNA sequence by translating the RNA complementary to pDd 812 in a heterologous system. The mRNA directs the synthesis of a major polypeptide of 33,000 daltons and a minor polypeptide of 31,000 daltons. We have used the plasmid DNA immobilized on filters to analyze the transcription of this RNA sequence in isolated nuclei. The amount of transcript synthesized in nuclei isolated at various stages of development which was complementary to pDd 812 changed in the same way as did the cytoplasmic concentration of this RNA--that is, maximal transcription occurred after 3-4 hr of development. Because this result was observed using labeling periods as short as 5 min, we believe that this change is unlikely to reflect a change in the rate of processing of RNA. We interpret these results to indicate that, at least in part, the control of the synthesis of this RNA is at the level of gene transcription.

Base Sequence↗
PubMed2001

Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

Animals↗
PubMed2000

Transcription and replication of the influenza a virus genome.

The genome of influenza A virus consists of eight segments of negative-strand viral RNA (vRNA). During the replication cycle of the virus, the genomic vRNA is transcribed into positive-strand mRNA and complementary RNA (cRNA) in the cell nucleus. The promoter for the synthesis of mRNA molecules is located in a partially double-stranded RNA structure formed by the 5'- and 3'-terminal sequences of genomic vRNA segments. The virus encoded RNA-dependent RNA polymerase complex has to interact with both ends of the vRNA in order to generate capped RNA primers by endonucleolytic cleavage of cellular pre-mRNAs for the initiation of viral mRNA synthesis. Conserved sequence elements in the 5'-end, e.g. a polymerase binding site and a U(5-7) sequence are required for polyadenylation of virus-specific mRNAs. Polyadenylation occurs by reiterative copying of the U(5-7) sequence by the viral RNA polymerase, which is bound to the 5'end of the vRNA template. The U(5-7) sequence acts directly as a template for the poly(A)-tail. During the replication cycle of the virus, a "switch" from mRNA to cRNA synthesis occurs, but the mechanism by which this switch occurs remains unclear. The viral nucleoprotein and its interaction with the polymerase proteins and vRNA might play a role in this process. In contrast to transcription, the process of replication--the synthesis of cRNA and vRNA, which are known to occur in the absence of primers--is poorly understood.

Animals↗
PubMed1995

Age-related alterations in the expression of prohormone convertase messenger ribonucleic acid (mRNA) levels in hypothalamic proopiomelanocortin mRNA neurons in the female C57BL/6J mouse.

POMC processing is mediated by the prohormone convertases (PC1 and PC2). The cleavage of beta-endorphin-(1-31) is mediated by PC2. PC2 can also further cleave beta-endorphin-(1-31) to beta-endorphin-(1-27). We previously reported a significant increase in the proportion of beta-endorphin-(1-27) and -(1-27) forms in the arcuate nucleus (ARC) of the hypothalamus in middle-aged females with irregular estrous cycles (5-7 days) compared to young female C57BL/6J mice with regular cycles (4-5 days). Changes in processing enzymes may be a mechanism underlying this change. We compared ARC messenger RNA (mRNA) levels of PC1, PC2, and furin by Northern blot and in situ hybridization analyses in young, middle-aged, and old mice. Antisense complementary RNA probes to mouse PC1, PC2, and furin were radiolabeled and used in single label studies, alone or in combination with a mouse POMC digoxigenin-labeled complementary RNA probe for double label studies. For Northern blot analysis, young (4- to 5-month-old) normally cycling (4-5 days) mice at diestrus were compared to middle-aged (12- to 13-month-old) irregularly cycling (5-7 days) mice at diestrus. By Northern blot analysis, a significant increase (P < 0.05) in ARC PC2 mRNA levels was detected in middle-aged compared to young mice, but ARC PC1 and furin mRNA levels were unaltered. Single label in situ hybridization analysis confirmed these findings in the general neuron population. We also observed a significant reduction in ARC furin mRNA levels in old mice compared to either young or middle-aged mice. Double labeling in situ hybridization histochemistry demonstrated that PC2 mRNA levels were significantly increased (at least 2-fold) in POMC mRNA-containing neurons of middle-aged compared to young mice. Selective changes in PC2 mRNA levels in ARC POMC neurons are correlated with changes in beta-endorphin-(1-31) processing to beta-endorphin-(1-27)/(1-26) in middle-aged animals. Our data suggest that the natural age-related shift in beta-endorphin peptide processing is mediated by PC2.

Aging↗
PubMed2004

Nonuniform hybridization: a potential source of error in oligonucleotide-chip experiments with low amounts of starting material.

Low amounts of starting material are a significant limitation of gene-expression profiling of microprepared pathologic specimens. Linear RNA amplification has become the method of choice to overcome this problem. Thus, transcriptomal analyses by oligonucleotide-chips or cDNA microarrays are now feasible with labeled complementary RNA generated from total RNA samples in the lower nanogram range. However, in case of oligonucleotide-chips, it has been underestimated so far that individual complementary RNA molecules are shorter in length than and display a 3' bias in comparison to the sequence stretch represented by oligonucleotides on the chip. This can lead to incorrect interpretation of raw data. We have analyzed this problem testing ex vivo-microprepared endothelial cells with Affymetrix GeneChips U133A. Only a small subset of housekeeping genes showed adequate uniform hybridization. We developed a software tool for objective evaluation of oligonucleotide-chips based on automated analysis of as well as normalization to this subset of housekeeping genes. We analyzed the gene expression profile of microprepared lymphatic vascular endothelial cells. We show that optimized normalization prevented exclusion of angiopoietin-2, a lymphatic endothelial marker, from the lymphovascular transcriptome.

Endothelial Cells↗
PubMed1993

Effect of an activin A on follicle-stimulating hormone (FSH) receptor messenger ribonucleic acid levels and FSH receptor expressions in cultured rat granulosa cells.

Activin, a dimer of beta-subunits of inhibin, has been found to induce FSH receptor on cultured rat granulosa cells. The effect of activin on FSH receptor messenger RNA (mRNA) levels has not been elucidated. To study the effect of activin on FSH receptor mRNA levels, we used a specific complementary RNA probe to evaluate changes in FSH receptor transcripts in cultured rat granulosa cells. Granulosa cells obtained from immature diethylstilbestrol-treated rats contained two predominant FSH receptor mRNA transcripts (5.5 and 2.4 kilobases). Compared to the control, the treatment of granulosa cells with activin (100 ng/ml) increased FSH receptor mRNA in a time-dependent manner with a maximum of about a 4-fold increase at 24 h. FSH receptor mRNA markedly decreased after 48 h and maintained a level comparable to that found in the control. The FSH receptor expression was also increased by activin. Scatchard analysis of the binding of rat FSH to granulosa cells showed that the increase in FSH binding after activin treatment was due to an increase in the receptor number and not the affinity of binding. Treatment of granulosa cells for 24 h with activin (20-300 ng/ml) increased FSH receptor mRNA in a dose-dependent manner to a maximum of about a 4-fold increase at a concentration of 100-300 ng/ml. We analyzed rat type II activin receptor mRNA transcripts in cultured rat granulosa cells with a specific complementary RNA probe to study the action of activin on granulosa cells. Granulosa cells contained two predominant rat type II activin receptor mRNA transcripts (6.0 and 3.0 kilobases). Furthermore, we measured intracellular cAMP production by activin to examine the mechanism by which activin acts on granulosa cells. In result, activin alone did not increase intracellular cAMP accumulation. In conclusion, this study demonstrates that the effect of activin A on the induction of FSH receptor expression is associated with a change in FSH receptor mRNA levels, suggesting that modulation of follicle development occurs.

Activins↗
PubMed1975

Influenza virion RNA-dependent RNA polymerase: stimulation by guanosine and related compounds.

The activity of RNA-dependent RNA polymerase of several influenza viruses is stimulated by guanosine. Depending upon the virus strain used, the stimulation of initial reaction rate is up to 10-fold. 5'-GMP, 3',5'-cyclic GMP, and 5'-GDP show lesser stimulation effects. No other nucleosides of 5'-NMPs stimulate, but the dinucleoside monophosphates GpG and GpC show large stimulations. We present evidence that the stimulation represents preferential initiation of genome complementary RNA chains with guanosine: (i) [3-H] guanosine is incorporated specifically at the 5'terminus of RNA in polymerase reaction mixes in vitro. (ii) This incorporation reaction has several properties similar to those of the virion polymerase elongation reaction. (iii) RNA made in the stimulated reaction behaves as complementary RNA in annealing kinetic studies, as does RNA labeled with [3-H]guanosine.

Cyclic GMP↗
PubMed1984

Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.

A library of double-stranded cDNA was constructed from ts13 cells, a G1-specific temperature-sensitive hamster cell line. The cDNAs, cloned into pBR322, were prepared from poly(A)+ mRNA isolated from ts13 cells 6 hr after serum stimulation at the permissive temperature of 34 degrees C. Differential screening of the library with G1-specific and G0-specific single-stranded cDNA probes prepared from the same cells identified five cDNA clones whose sequences were preferentially expressed in G1. Levels of RNA complementary to these clones were 3- to 6-fold higher in G1 than in other phases of the cell cycle. When ts13 cells were arrested in G1 at the restrictive temperature of 39.6 degrees C, the levels of RNA complementary to p13-2A9 and p13-4F1 were as high as 10 times that found in a resting population, while the expression of sequences complementary to p13-2A8 did not significantly change from levels found in G0. RNA and Southern gel blot analysis suggest that these cell-cycle-specific clones represent either low copy or moderately repetitive gene sequences. Results with another ts mutant of the cell cycle, tsAF8, which is a ts mutant of RNA polymerase II, showed that these cell-cycle-specific sequences have a rapid turnover. The use of G1-specific ts mutants of the cell cycle provides an approach to determine which cell-cycle-dependent genes are most relevant to cell cycle progression.

Animals↗
PubMed2003

Synthesis and properties of 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) as effective antisense oligonucleotides.

Novel bicyclo nucleosides, 2'-O,4'-C-ethylene nucleosides and 2'-O,4'-C-propylene nucleosides, were synthesized as building blocks for antisense oligonucleotides to further optimize the 2'-O,4'-C-methylene-linkage of bridged nucleic acids (2',4'-BNA) or locked nucleic acids (LNA). Both the 2'-O,4'-C-ethylene- and propylene-linkage within these nucleosides restrict the sugar puckering to the N-conformation of RNA as do 2',4'-BNA/LNA. Furthermore, ethylene-bridged nucleic acids (ENA) having 2'-O,4'-C-ethylene nucleosides had considerably increased the affinity to complementary RNA, and were as high as that of 2',4'-BNA/LNA (DeltaT(m)=+3 approximately 5 degrees C per modification). On the other hand, addition of 2'-O,4'-C-propylene modifications in oligonucleotides led to a decrease in the affinity to complementary RNA. As for the stability against nucleases, incorporation of one 2'-O,4'-C-ethylene or one 2'-O,4'-C-propylene nucleoside into oligonucleotides considerably increased their resistance against exonucleases to an extent greater than 2',4'-BNA/LNA. These results indicate that ENA is more suitable as an antisense oligonucleotide and is expected to have better antisense activity than 2',4'-BNA/LNA.

Alkenes↗
PubMed2000

Solution structure of a HNA-RNA hybrid.

BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.

Base Sequence↗
PubMed1999

Paradoxes of the replication of picornaviral genomes.

A wealth of experimental data on the mechanism of the picornavirus genome replication has accumulated. Not infrequently, however, conclusions derived from these data appear to contradict each other. On the one hand, initiation of a complementary RNA strand can be demonstrated to occur in a solution containing only the poliovirus RNA polymerase, VPg, uridine triphosphate, poly(A) template and appropriate ions. On the other hand, convincing experiments suggest that efficient initiation of a viral complementary RNA strand requires complex cis-acting signals on the viral RNA template, additional viral and possibly cellular proteins as well as a membrane-containing environment. On the one hand, there is evidence that the viral RNA, in order to be replicated, should first be translated, but on the other hand, the viral RNA polymerase appears to be unable to overcome the ribosome barrier. Possible solutions for these and several other similar paradoxes are discussed, along with less contradictory results on the properties of the picornaviral replicative proteins. Recent results suggesting that recombination and other rearrangements of the viral RNA genomes may be accomplished not only by the replicative template switching but also by nonreplicative mechanisms are also briefly reviewed.

Genome, Viral↗
PubMed1982

Antibody to a host protein prevents initiation by the poliovirus replicase.

In vitro transcription of poliovirus RNA was catalyzed by the combination of a virus-coded polymerase and a host cell protein (host factor). Antibody to host factor inhibited template-dependent synthesis of complementary RNA where presumably RNA chain initiation occurred. On the contrary, elongation of already initiated RNA chains catalyzed by the replicase-template complex was not inhibited by anti-host factor antibody. These results strongly favored our previous notion that the host factor was needed for the initiation step of viral complementary RNA synthesis.

Antibodies↗
PubMed1996

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Amino Acid Sequence↗
PubMed1972

Molecular hybridization of ribonucleic acid with a large excess of deoxyribonucleic acid.

When RNA is annealed in solution with a sufficiently large excess of DNA, the kinetics of DNA-RNA hybridization are relatively simple. Methods are described for following the course of both DNA renaturation and DNA-RNA hybridization in this system. To explore the characteristics of the reaction a series of model systems was used. Each one utilized DNA (sheared to constant size) from a bacterium or bacteriophage and homologous cRNA, i.e. RNA synthesized in vitro on a template of the same DNA. Temperature optima were determined for the hybridization of Escherichia coli nucleic acids in 2xSSC and 3xSSC-50% formamide buffers, and of Proteus mirabilis nucleic acids in 2xSSC buffer. Rate-constants for DNA-RNA hybridization were measured by two methods. These gave somewhat different results, but in all cases the rate-constant of DNA-RNA hybridization was clearly less than that of DNA renaturation. Thus hybridization is a slower reaction than DNA renaturation. Nevertheless, in some cases, with a high concentration of DNA and a long annealing time, 90-95% of the added RNA became resistant to ribonuclease. Experiments are described which show that it is possible to deduce the analytical complexity of DNA with reasonable accuracy from its hybridization with complementary RNA. Similarly, it is possible to estimate the reiteration frequency of multiple DNA sequences (such as ribosomal DNA) from the hybridization of the total DNA with RNA complementary to the multiple sequences. The effect on the system of various DNA/RNA ratios from 100 to 1 is described.

Coliphages↗
PubMed1998

Novel mutations in aquaporin-2 gene in female siblings with nephrogenic diabetes insipidus: evidence of disrupted water channel function.

Novel mutations of the aquaporin-2 (AQP2) gene have been detected in Japanese female siblings with autosomal-recessive nephrogenic diabetes insipidus. The patients were compound heterozygote for point mutations at nucleotide position 374 (C374T) and at position 523 (G523A) in exon 2 of the AQP2 gene, resulting in substitution of methionine for threonine at codon 125 (T125M) and arginine for glycine at codon 175 (G175R). The water permeability (Pf) of oocytes injected with wild-type complementary RNA increased 9.0-fold compared with the Pf of water-injected oocytes, whereas the increases in the Pf of oocytes injected with T125M and G175R complementary RNA were only 1.7-fold and 1.5-fold, respectively. Immunoblot and immunocytochemistry indicated that the plasma membrane expressions of T125M and G175R AQP2 proteins were comparable to that of the wild-type, suggesting that although neither the T125M nor G175R mutation had a significant effect on plasma membrane expression, they both distorted the structure and function of the aqueous pore of AQP2. These results provide evidence that the nephrogenic diabetes insipidus in patients with T125M and G175R mutations is attributable not to the misrouting of AQP2, but to the disrupted water channel function.

Amino Acid Sequence↗
PubMed1998

[Peptide nucleic acids and their phosphonate analogues: synthesis and hybridization characteristics].

The synthesis of a series of DNA mimics--peptide nucleic acids, phosphonate analogues of peptide nucleic acids, and their hybrids--is described. The preparative synthesis of the corresponding monomers and the solid phase automated synthesis of oligomers-mimics are developed. Modified phosphonate analogues of peptide nucleic acids, in particular chiral derivatives and those with additional hydroxyl groups in the side chains of the backbone as well as pyrene derivatives of peptide nucleic acids and their phosphonate analogues, are prepared. The ability of the resulting oligomers specifically to hybridize to DNA and RNA complementary chains is studied. It is shown that phosphonate analogues of peptide nucleic acids and their hybrids with peptide nucleic acids can form complexes with the DNA and RNA complementary strands, the stability of the complexes increasing in parallel with the increase in the number of peptide nucleic acid residues in the chain of the mimic. This property, along with good water solubility, provides the precondition for further evaluation of these compounds as antisense and antigene agents.

Chromatography, High Pressure Liquid↗