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Results for “RNA, Complementary”

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PubMed2000

Crystal structure of the bacterial conjugation repressor finO.

The conjugative transfer of F-like plasmids is repressed by FinO, an RNA binding protein. FinO interacts with the F-plasmid encoded traJ mRNA and its antisense RNA, FinP, stabilizing FinP against endonucleolytic degradation and facilitating sense-antisense RNA recognition. Here we present the 2.0 A resolution X-ray crystal structure of FinO, lacking its flexible N-terminal extension. FinO adopts a novel, elongated, largely helical conformation. An N-terminal region, previously shown to contact RNA, forms a positively charged alpha-helix (helix 1) that protrudes 45 A from the central core of FinO. A C-terminal region of FinO that is implicated in RNA interactions also extends out from the central body of the protein, adopting a helical conformation and packing against the base of the N-terminal helix. A highly positively charged patch on the surface of the FinO core may present another RNA binding surface. The results of an in vitro RNA duplexing assay demonstrate that the flexible N-terminal region of FinO plays a key role in FinP-traJ RNA recognition, and supports our proposal that this region and the N-terminus of helix 1 interact with and stabilize paired, complementary RNA loops in a kissing complex.

Amino Acid Sequence↗

PubMed1997

Transplatin-modified oligo(2'-O-methyl ribonucleotide)s: a new tool for selective modulation of gene expression.

In the reaction between trans-diamminedichloroplatinum(II) and single-stranded oligo(2'-O-methyl ribonucleotide)s containing the sequence GNG (N being a nucleotide residue), the 1,3-trans-{Pt-(NH3)2[GNG]} cross-links are formed. The 1,3-intrastrand cross-links are inert within the single-stranded oligonucleotides. By contrast, they rearrange into interstrand cross-links when the platinated oligonucleotides are paired with their complementary RNA strands. The rate of the interstrand cross-linking reaction depends upon the sequence facing the intrastrand cross-links. When the complementary sequences are 5'-CN'C (N' being a nucleotide), the rates are rather slow (T1/2 >/= 3 h at 37 degrees C). The rearrangement of the intrastrand cross-links into interstrand cross-links can be achieved in a few minutes when the triplets facing the intrastrand cross-links are replaced by doublet 5'-UA or 5'-CA. In vitro, the specificity of the cross-linking reaction between a platinated oligo(2'-O-methyl ribonucleotide) and its target sequence (containing the 5'-CA doublet) located within the coding region of Ha-ras mRNA is demonstrated by steric blocking of reverse transcriptase and translation machinery. Within the HBL100ras1 cells, this platinated oligonucleotide binds specifically and irreversibly to the cognate Ha-ras mRNA. It also inhibits the proliferation of the HBL100ras1 cells in a dose-dependent manner. The fast and specific interstrand cross-linking reaction triggered by the formation of a double helix between platinated oligo(2'-O-methyl ribonucleotide)s and RNA enhances the potential of the oligonucleotides which do not induce mRNA cleavage by RNase H, to modulate gene expression by steric blocking of the translation machinery.

Cell Division↗

PubMed1996

In Xenopus oocytes the human C3a and C5a receptors elicit a promiscuous response to the anaphylatoxins.

The Xenopus laevis oocyte has been widely utilized for cloning and functional expression of G-protein coupled receptors (GPCR). This system was used for the functional expression and characterization of the recently identified human C3a receptor. Complementary RNA from the human C3a receptor was transcribed in vitro and microinjected into Xenopus oocytes for functional characterization. A positive response to a synthetic C3a peptide agonist and to C3a, but not to platelet activating factor or fMetLeuPhe was detected. In addition, a response of approximately one third the amplitude obtained with C3a was obtained with rC5a. Conversely, oocytes co-injected with the C5a receptor and total RNA isolated from U937 cells responded to C5a as well as to C3a and the C3a synthetic peptide. A functional response with the anaphylatoxin C3a receptor in oocytes was dependent on co-injection of a pertussis toxin sensitive complementary human factor which could be supplied by co-injection of total RNA isolated from U937 cells. Oocytes expressing the anaphylatoxin C3a and C5a receptors responded to both agonists, in each case the response to the cognate ligand was substantially more robust than the response elicited by the other anaphylatoxin.

Amino Acid Sequence↗

PubMed2001

Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines.

Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis. In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site. We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA. The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out. Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing. In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions.

Adenosine↗

PubMed1994

Detection of interleukin-5 messenger RNA and interleukin-5 protein in bronchial biopsies from asthma by nonradioactive in situ hybridization and immunohistochemistry.

Recently direct evidence for the role of interleukin-5 (IL-5) in eosinophilic inflammation in the airways of persons with asthma has been provided by an in situ hybridization study that used radioisotope-labeled IL-5 complementary RNA probes. Radioisotope-labeled probes, although sensitive, require autoradiographic detection, which is time-consuming. In the most recent study we attempted to detect IL-5 messenger RNA in the bronchial biopsy specimens from patients with asthma using nonradioactive in situ hybridization, which gives rapid results. Bronchial biopsy specimens were obtained from eight patients with asthma and seven diseased control subjects. IL-5 complementary DNA probes were labeled with digoxigenin-deoxyuridine triphosphate and hybridized to permeabilized sections. Hybridization signals were visualized by an immunohistochemistry technique. Positive hybridization signals were observed in six of the eight biopsy specimens from patients with asthma. Pretreatment with ribonuclease or hybridization with an unrelated probe produced negative results. Immunohistochemical staining of serial sections with a monoclonal antibody to IL-5 revealed that a few cells within the mucosa positively stained, suggesting active synthesis of IL-5. Biopsy results from the seven diseased control subjects did not show any hybridization signal. These results confirm and extend previous observations of IL-5 messenger RNA expression in the airways of patients with asthma, and suggest that digoxigenin-labeled IL-5 complementary DNA probes would be a powerful research tool.

Adult↗

PubMed1995

Glucocorticoids stimulate thyrotropin-releasing hormone gene expression in cultured hypothalamic neurons.

Although there is much evidence indicating that glucocorticoids (GC) inhibit the hypothalamic-pituitary-thyroid axis in both rat and man in vivo, there have been no previous studies on the direct effect of GC on hypothalamic TRH neurons in vitro. In this laboratory, we developed fetal rat (day 17) diencephalic neuronal cultures in the presence of 5'-bromo-2-deoxyuridine, a cell-differentiating agent that stimulates TRH gene expression. In 12 separate experiments, dexamethasone (Dex) induced a 2.2-fold increase in TRH content vs. the control value (P < 0.01). Dex (10(-8)M) enhanced TRH messenger RNA (mRNA) 1.6-fold (n = 75 wells; P < 0.01) by nonisotopic in situ hybridization. On Northern blot analysis using a 32P-labeled complementary RNA probe, TRH mRNA was enhanced 3-fold (n = 4; P < 0.01). Nuclear run-on analysis revealed that Dex enhanced transcription 7.7 fold (n = 3; P < 0.01). We conclude that 1) Dex stimulates the expression of TRH peptide and TRH mRNA in cultured hypothalamic neurons; 2) the increase in TRH mRNA results (at least in part) from enhanced transcription; and 3) the reported in vivo depression of TRH in the paraventricular nucleus after GC stimulation suggests that this effect must be mediated indirectly on the TRH neuron.

Animals↗

PubMed1997

In situ hybridization histochemical localization of prodynorphin messenger RNA in the central nervous system of the rat.

The distribution of preprodynorphin messenger RNA-containing perikarya in the central nervous system of the rat was determined with in situ hybridization histochemistry using a 35S-labelled complementary RNA probe. All of the regions of the central nervous system reported by other investigators to contain perikarya that synthesize prodynorphin-derived peptides, except the pedunculopontine tegmental nucleus, the accessory trigeminal nucleus, and the ventral nucleus of the trapezoid body, also contained perikarya that synthesize preprodynorphin messenger RNA. However, the olfactory bulb, the anterior olfactory nucleus, the islands of Calleja, the CA1-CA3 fields of the hippocampus, the septohippocampal nucleus, the diagonal band of Broca, the basal and cortical amygdaloid nuclei, the entopeduncular nucleus, the subthalamic nucleus, the superior colliculus, the Edinger-Westphal nucleus, the dentate nucleus, the raphes linearis and pontis, the dorsal cochlear nucleus, the medial vestibular nucleus, the inferior olive, and the dorsal motor nucleus of the vagus nerve also contained preprodynorphin messenger RNA-synthesizing perikarya. These observations suggest that prodynorphin-derived peptides have a much more pervasive role in central nervous system function than previously suspected. However, before the physiological significance of these observations can be judged, it will be necessary to determine whether all of the novel sites of preprodynorphin messenger RNA synthesis are sites of prohormone synthesis and conventional processing.

Animals↗

PubMed2005

A surface-mediated origin of the RNA world: biogenic activities of clay-adsorbed RNA molecules.

The involvement of clay surfaces in the origin of the first genetic molecules on Earth has long been suggested. However, the formation of these polymers was not sufficient by itself to initiate the evolutionary process leading to the appearance of life. These macromolecules had to persist in primeval habitats so that their biological potentiality could be expressed. In this study, we assess the possibility of development of the RNA world on a clay substrate by investigating the capacity of different RNA molecules adsorbed/bound on the clay minerals montmorillonite (M) and kaolinite (K) to persist in the presence of a degrading agent (RNase-A), to interact specifically with complementary RNA strands, and to transmit the information contained in their nucleotide sequences. The RNase-A degradation of clay-adsorbed 23S rRNA from Escherichia coli was significantly slower (75-80%) than that observed for free rRNA, and the complete digestion of nucleic acid in the presence of clay was obtained in 2 vs. 1 h. Clay-adsorbed Poly[A] homopolymer was able to recognize the complementary Poly[U] homopolymer present in the surrounding water solution and to establish a specific interaction (association) with it, possibly leading to the formation of double strands. Reverse transcription and amplification (RT-PCR) amplification of free and clay-adsorbed 16S indicated that the presence of clay particles partially reduced the efficiency and processivity of reverse transcriptase but did not inhibit its activity, demonstrating that clay-adsorbed RNA is still available for enzymatic replication. These findings indicate that primordial genetic molecules adsorbed on clay minerals would have been protected against degrading agents present in the environment and would have been in the right conditions to undergo evolutionary processes.

Aluminum Silicates↗

PubMed1992

Complete sequence of maize stripe virus RNA4 and mapping of its subgenomic RNAs.

The complete nucleotide sequence of maize stripe virus RNA4 was determined and found to consist of 2227 nucleotides containing two significant open reading frames. One, in the 5' end of the viral RNA, encodes the major non-capsid protein of M(r) 19,815. The other is located in the 5' end of the viral complementary RNA and could encode a protein of M(r) 31,900. This protein has not been identified previously and has been designated NS4, a non-structural protein. RNA-RNA hybridization detected subgenomic RNAs encoding these proteins, a characteristic of RNA possessing an ambisense gene organization.

Amino Acid Sequence↗

PubMed1988

Constitutive expression of c-fos antisense RNA blocks c-fos gene induction by interferon and by phorbol ester and reduces c-myc expression in F9 embryonal carcinoma cells.

To address the role of c-fos proto-oncogene we constructed a plasmid that allows constitutive expression of RNA complementary to c-fos mRNA, and stably introduced this plasmid into F9 embryonal carcinoma cells. Some F9 clones expressing c-fos antisense RNA had a reduced basal level of c-fos mRNA, and were unable to induce a c-fos mRNA as well as its protein when stimulated with phorbol ester or with interferon (IFN). Nevertheless, the ability to induce major histocompatibility class I genes following IFN treatment was not impaired in these clones. Clones expressing c-fos antisense RNA grew as rapidly as control F9 cells, and underwent differentiation after retinoic acid treatment. Unexpectedly, constitutive expression of c-myc mRNA was reduced on average by 10-fold in clones expressing c-fos antisense RNA. However, expression of the p53 gene and heat shock gene hsp 70 was not affected in these clones, indicating the existence of a specific regulatory linkage between c-fos and c-myc genes. Cycloheximide treatment led to induction of a large amount of c-fos mRNA in clones expressing c-fos antisense RNA as well as in control F9 clones. The amount of c-fos antisense RNA was also increased by cycloheximide treatment. We postulate that c-fos antisense RNA blocks expression of the endogenous c-fos gene by accelerating the degradation of c-fos mRNA and that cycloheximide treatment interferes with this degradation.

Cell Differentiation↗

PubMed1993

Determination of estrogen receptor messenger ribonucleic acid (mRNA) and cytochrome P450 aromatase mRNA levels in adipocytes and adipose stromal cells by competitive polymerase chain reaction amplification.

Clinical evidence suggests that sex hormones affect adipose tissue metabolism and deposition. To investigate the biosynthesis and possible action of estrogen in adipose tissue, we report the use of competitive, specific polymerase chain reaction amplifications to determine levels of estrogen receptor (ER) messenger RNA (mRNA) and cytochrome P450 aromatase mRNA in adipocytes and adipose stromal cells. This extremely sensitive technique uses coamplification of a homologous animal species complementary RNA to control for differences in amplification efficiencies. The DNA amplification products are identified by Southern hybridization with species-specific radiolabeled oligonucleotide probes. Abdominal adipose tissue obtained from female patients during elective abdominoplasty was separated by collagenase digestion and centrifugation into floating adipocytes and pelleted adipose stromal cells. Our results demonstrate higher ER mRNA levels in adipocytes compared to adipose stromal cells, whereas cytochrome P450 aromatase mRNA levels are higher in adipose stromal cells compared to adipocytes. The finding of ER mRNA in adipose tissue suggests the presence of the ER in adipose tissue. In addition the inverse correlation of ER mRNA and cytochrome P450 aromatase mRNA levels in adipocytes and adipose stromal cells suggests a paracrine relationship whereby estrogen produced by adipose stromal cells affects adjacent adipocytes.

Adipocytes↗

PubMed1991

Synaptic activity results in increased transcription as detected by the brain-specific probe pEL-48 in Torpedo marmorata.

The cloning of sequences expressed exclusively in neurons that use acetylcholine as a neurotransmitter resulted in multiple probes. One of them, pEL-48, was used to detect changes in transcription patterns in the electric lobes of Torpedo marmorata. Fish were stimulated either electrically or mechanically to induce discharge of the electric organs; they were killed after different poststimulation time intervals. The RNA isolated from the electric lobes was quantified using Northern blots and pEL-48 as a probe; distinct changes in the quantity of pEL-48 complementary RNA could be detected. Partial sequencing of the clone pEL-48 revealed an unusual primary sequence with numerous short open reading frames, coding for hitherto unknown polypeptides.

Acetylcholine↗

PubMed1985

Intermolecular exon ligation of the rRNA precursor of Tetrahymena: oligonucleotides can function as 5' exons.

The dinucleotide CpUOH, when incubated with self-splicing Tetrahymena pre-rRNA in the absence of GTP, functions as a 5' exon. It cleaves the precursor exactly at the 3' splice site and becomes covalently ligated to the 3' exon. Other oligonucleotides with sequences that resemble CUCUCU, the sequence at the 3' end of the 5' exon, can add to the 3' exon in this reaction. Such splicing in trans is most readily explained by a site within the intervening sequence that binds the last few nucleotides of the 5' exon. This binding site functions in splice site recognition and is also part of the active site of the ribozyme. The mechanism by which 5' splice sites are selected in Tetrahymena rRNA and group I mitochondrial RNA splicing is like that used in nuclear mRNA splicing, in that it involves specific pairing of bases adjacent to the splice site with a complementary RNA sequence.

Animals↗

PubMedDate not supplied

Aminomodified nucleobases: functionalized nucleoside triphosphates applicable for SELEX.

5-Aminoallyl-2'-fluoro-dUTP, 5-aminoallyl-UTP, and N(6)-([6-aminohexyl]carbamoylmethyl)-ATP were systematically tested for their suitability for the systematic evolution of ligands by exponential enrichment (SELEX) process with the aim of introducing additional functionalities to RNA libraries. All three aminomodified nucleoside triphosphates proved to be compatible with the enzymatic steps required for SELEX and maintained strict Watson-Crick basepairing. Complementary RNA molecules modified with the two uridine analogues show a significantly increased melting temperature, whereas the introduction of N(6)-([6-aminohexyl]carbamoylmethyl)-ATP leads to a decreased T(m) and thus less stable basepairing. The chemical synthesis of 5-aminoallyl-2'-fluoro-dUTP is reported in detail.

Adenosine Triphosphate↗

PubMed1979

Regulation of the primary expression of the early adenovirus transcription units.

The time course of appearance of transcriptional activity from five early adenovirus type 2 transcription units has been determined. RNA complementary to region 1A (1-4.4 map units), the first region to be transcribed, was detectable at 45 min after infection; a maximal rate of RNA synthesis was reached at 3 h after infection and was maintained thereafter for at least 6 h. RNA from region 2 (75-56 map units), which encodes the mRNA for the 72,000-dalton DNA-binding protein, was the last to be synthesized; transcription commenced at about 2 h postinfection, reached a maximum at 7 h, and then declined. Transcription of regions 3 (76-86 map units) and 4 (99-91 map units) reached a maximal value at 3 h postinfection. The rates of RNA synthesis from these regions then declined over the next 6 h. The decline of transcription from regions 2 and 4 appeared to be a specific repression of these transcription units. The repression did not occur in the absence of protein synthesis, suggesting that a viral protein might be involved. Transcription of all early regions was initiated and continued for at least 2 to 3 h in cells that were treated with cycloheximide or emetine before and during infection, suggesting that at least the initiation of RNA synthesis from the five early adenovirus type 2 transcription units does not depend on the formation of a viral protein. Moreover, mRNA was formed in the absence of protein synthesis that hybridized to DNA fragments representing each of the five early transcription units. The increase in mRNA accumulation in the presence of cycloheximide (or emetine) does not appear to be due to increased RNA synthesis; thus, either increased mRNA stability or increased efficiency of nuclear RNA processing must occur.

Adenoviruses, Human↗

PubMed1990

Glucocorticoid receptor messenger ribonucleic acid in different regions of human adipose tissue.

The expression of glucocorticoid receptor (GR) messenger RNA (mRNA) was investigated in sc adipose tissue and isolated adipocytes from the abdominal and gluteal regions in men and women using a human GR complementary RNA probe. GR mRNA levels were 2-fold higher in female than in male abdominal tissue or adipocytes, whereas in gluteal tissue or adipocytes no sex differences were observed. GR mRNA levels in female abdominal adipocytes were 50% higher than in corresponding female gluteal adipocytes; the opposite was observed corresponding in males. Northern blot analysis of total cellular RNA isolated from abdominal and gluteal adipocytes showed hybridization of the human GR probe to an RNA species of approximately 7.1 kilobases in both regions. No sex or regional differences in GR mRNA stability were observed. The human metallothionein II (hMTII) mRNA, which is regulated by glucocorticoids at the transcriptional level, showed an opposite sex and regional pattern as GR mRNA. However, in gluteal adipose tissue no sex differences were observed in hMTII mRNA levels. The expression of beta-actin mRNA, which is not regulated by glucocorticoids, showed no sex or regional variation. By immunocytochemistry, using an anti-GR-monoclonal antibody, cytoplasmic as well as nuclear staining for GR was demonstrated in both sexes and both regions. In conclusion, variations in GR mRNA levels between sexes and body regions may explain the well known sex and tissue differences in effects of glucocorticoids on human adipose tissue.

Abdomen↗

PubMed1997

[Transcription and replication of influenza virus genome].

The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vRNA) segment and after infection, involved in both transcription (vRNA-directed synthesis of viral mRNA) and vRNA replication (vRNA-dependent synthesis of complementary RNA(cRNA) and cRNA-dependent synthesis of vRNA). The RNA polymerase is composed of three viral proteins, PB1, PB2 and PA. PB1 is the core subunit for not only the RNA synthesis but also the assembly of PB2 and PA into this multifunctional enzyme complex. PB1 alone is able to catalyze vRNA-dependent RNA synthesis, but PB2 is required for capped RNA-dependent transcription, both together forming the transcriptase. The third P protein, PA, and an as yet unidentified host factor(s) are involved for the conversion of RNA polymerase from transcriptase to replicase. The functional map including both subunit-subunit contact sites and catalytic sites for capped RNA endonuclease and RNA polymerization is being made for both PB1 and PB2 proteins.

Genome, Viral↗

PubMedDate not supplied

Expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the human fetal adrenal.

OBJECTIVE: To understand better the steroidogenic capacity of the human fetal adrenal (HFA), we evaluated the expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the fetal zone and neocortex of the HFA using a specific RNase protection assay. METHODS: Adrenal glands were obtained at the time of elective termination of pregnancy. Whole adrenals (n = 7) were frozen in liquid nitrogen, and subsequently total RNA extraction was performed by tissue homogenization followed by guanidinium/chloroform purification. In addition, RNA was obtained from separated fetal zone (n = 4) and neocortex (n = 4) tissues obtained by dissection. RNase protection assays were then performed using radiolabeled complementary RNA probes generated by T7 RNA polymerase directed against transcripts for CYP11B1, CYP11B2, and actin, the latter of which was used as a control for RNA integrity. Transcripts also were examined using a reverse transcription polymerase chain reaction (RT-PCR) protocol specific for CYP11B1 or CYP11B2. RESULTS: The RNase protection assay was designed to distinguish specific bands that corresponded to CYP11B1 (232 bp), CYP11B2 (262 bp), and actin (221 bp). RNA isolated from whole HFA was observed to have high levels of CYP11B1 transcript, whereas CYP11B2 was not detected. Dissected neocortex and fetal zones were found to contain transcript for CYP11B1 using both the RNase protection assay and RT-PCR analysis. In contrast, using the RNase protection assay, CYP11B2 mRNA was not observed in the RNA from the fetal zone, but after prolonged exposure there was a band corresponding in size to CYP11B2 observed in RNA from the neocortex. Using the more sensitive RT-PCR method, transcript for CYP11B2 was found in both neocortex and fetal zone. CONCLUSION: The HFA expresses low levels of CYP11B2 in accordance with its low production of mineralocorticoid. The expression of CYP11B1 in the fetal zone is intriguing because this enzyme is not necessary for the production of C19 steroids. Definition of the molecular mechanisms controlling expression of the CYP11B genes will be necessary to determine why the HFA differentially expresses these isoenzymes.

Actins↗