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PubMed1979

Molecular cytogenetics of the Equidae. I. Purification and cytological localization of a (G + C)-rich satellite DNA from Equus przewalskii.

A (G + C)-rich density satellite DNA (rho = 1.713 gm/cc) has been purified from splenic DNA of Przewalski's horse, Equus przewalskii, by successive equilibrium density gradient centrifugations. The purified satellite, which may comprise as much as 29% of the total DNA, renatures rapidly; however, analyses of native, single-stranded, and reassociated molecules by analytical ultracentrifugation and melting properties suggest that some sequence heterogeniety exists in the 1.713 gm/cc satellite. Complementary RNA (cRNA) transcribed from satellite DNA has been utilized for in situ hybridization studies with E. przewalskii metaphase chromosomes previously identified by quinacrine-banding. These studies establish that sequences complementary to the 1.713 g/cc satellite are greatly enriched in the centromeres of some, but not all, chromosomes. The differential distribution of satellite DNA sequences over heterochromatic regions allows discrimination of three classes of heterochromatin and serves to define three types of pericentromeric regions in the karyotype of this endangered equine species. Additionally, apparent polymorphism in concentrations of satellite DNA sequences between homologs in the same karyotype is noted.

Animals↗

PubMed2002

Internal initiation by the cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences.

Tombusviruses, small positive sense RNA viruses of plants, are replicated by the viral-coded RNA-dependent RNA polymerase (RdRp) in infected cells. An unusual feature of the tombusvirus RdRp that is partially purified from cucumber necrosis virus (CNV)-infected plants is the ability to initiate complementary RNA synthesis from several internal positions on minus-strand templates derived from DI RNAs ( Nagy and Pogany, 2000 ). In this study, we used template deletion, mutagenesis, and oligo-based inhibition of RNA synthesis to map the internal initiation sites observed with the in vitro CNV RdRp system. Comparing sequences around the internal initiation sites reveals that they have either (i) similar sequences to the core minus-strand initiation promoter; or (ii) similar structures to the core plus-strand initiation promoter. In addition, we find similarities among the internal initiation sites and the subgenomic RNA initiation sites. These similarities suggest that the mechanism of internal initiation is similar to initiation from the terminal core promoters or the putative subgenomic promoter sequences. We propose that internal initiation on full-length RNA templates may be important in defective interfering (DI) RNA formation/evolution by producing intermediate templates for RNA recombination in tombusviruses. This may explain why tombusviruses are frequently associated with DI RNAs.

Base Sequence↗

PubMed1993

Amplification and detection of a single molecule of human immunodeficiency virus RNA.

Detection of plasma viremia in human immunodeficiency virus type 1 (HIV-1)-infected people is indispensable for the diagnosis of seronegative infection as well as for the evaluation of virus activities in vivo. The direct detection of HIV-1 RNA in circulation has been performed by means of reverse transcription followed by polymerase chain reaction (RT-PCR). As an attempt to establish a highly sensitive assay, we evaluated the effects of two-step amplification with nested primers and double priming of reverse transcription on the sensitivity of RT-PCR. The sensitivity of two-step amplification was 100 times higher than that of one-step amplification. The double priming of reverse transcription further increased the sensitivity of the following two-step amplification 100 times, which appeared to be enough to detect HIV-1 RNA from as little as a 2.2 x 10(-4) TCID50 unit equivalent of culture supernatant of HIV-1-infected cells and a single molecule of HIV-1 gag complementary RNA synthesized by in vitro transcription. By use of this most sensitive assay, we successfully detected HIV-1 RNA in serum or plasma from all 22 patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) and 13 out of 14 untreated asymptomatic carriers. Of 43 asymptomatic carriers under the treatment with interferon-alpha or azidothymidine, 17 cases showed negative results, indicating that the virus activity was suppressed by the therapeutics. We also noted the inhibitory effect of heparin on RT-PCR.

Base Sequence↗

PubMed1999

Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.

Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing.

Fluorescein-5-isothiocyanate↗

PubMed2001

Synthesis of small multifunctional molecules having nucleic acid binding property.

We have previously reported that a multifunctional conjugate having the acridine derivative as an intercalative molecule and the polyamine moiety as an RNA cleaving molecule bound to a double helical RNA and cleaved the target efficiently. Along the study to develop a sequence and site specific artificial RNA cleaving molecule, we have development a novel intercalative molecule having polyamine moiety and DNA connecting function. The trisamine-acridine-tethered DNA is expected to bind to the complementary RNA and cleave the phosphodiester bond of the RNA near the position of the built-in interecalator.

Acridines↗

PubMed1972

Reovirus replicase-directed synthesis of double-stranded ribonucleic acid.

After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand ((3)H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of (3)H-cytidine triphosphate (or (3)H-uridine triphosphate) and adenine triphosphate-alpha-(32)P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3'-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of (3)H to (32)P in these fragments indicated that RNA synthesis proceeded from the 5' to 3' terminus.

Animals↗

PubMedDate not supplied

[Expression of the liver mitochondrial genome in diabetes].

In vivo labelled liver mitochondrial RNA of normal and alloxan-diabetic rats are hybridized with mitochondrial DNA with similar efficiency. The liver mitochondrial RNAs have equal ability to competition with N-specific complementary RNA for hybridization on mitochondrial DNA and its fragments cloned in the composition of bacterial plasmids and characterized by a definite set of genes. Differences between concurrent ability of the liver mitochondrial RNAs of normal and alloxan-diabetic animals were not observed. There were no electrophoretic distinctions in mitochondrial RNAs in both groups of animals during RNA electrophoresis in agaroso-acrylamide gel, as well. The results obtained indicate a close set of RNA molecules in the liver mitochondria of normal and diabetic rats. Thus, a previously observed RNA biosynthesis decrease in the liver mitochondria in diabetes is caused not by a selected inhibition of definite gene expression but by a gradual fall of transcription of all the mitochondrial genome parts.

Alloxan↗

PubMed1987

The S segment of the Germiston virus RNA genome can code for three proteins.

The complete sequence of the S segment of Germiston bunyavirus has been determined from plasmids containing S cDNA inserts. The S segment is 980 nucleotides long with the first 15 bases at the 3' end complementary to the first 15 bases at the 5' end. Three overlapping open reading frames (ORF) were identified in the viral complementary RNA strand. The first ORF codes for a polypeptide of 233 amino acids (Mr 26,600) which is the nucleoprotein N. The second ORF codes for a polypeptide of 109 amino acids (Mr 11,800) which corresponds to the NSS protein, also called p12. Following this ORF, in the same frame, a third ORF which could encode a polypeptide of 75 amino acids was identified. Such a polypeptide has not yet been detected in infected cells. The N and NSS proteins of Germiston virus were compared with the corresponding proteins of La Crosse, snowshoe hare, and Aino viruses, and show a high extent of homology.

Amino Acid Sequence↗

PubMed1979

Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2mu circle.

We have identified two major and approximately ten minor poly(A)-containing RNA species in S. cerevisiae which arise from in vivo transcription of the yeast plasmid, known as 2mu circle. The two major species, which are 1325 and 1275 bases in length, are transcribed from the two unique halves of the plasmid and extend into the inverted repeat sequences which separate the unique regions. The map positions of the minor transcripts, which range in length from 350 to 2600 bases, indicate that except for a small region of the genome in which no transcription is observed, both strands of the entire 2mu circle genome are transcribed. We also present evidence demonstrating that RNA transcribed from 2mu circular DNA is used to program the synthesis of specific proteins in yeast: that is, yeast RNA complementary to 2mu circle DNA can be translated in vitro to produce specific polypeptides of substantial size. Finally, the pattern of transcription of 2mu circle suggests the possibility that messenger RNA species are derived by cleavage of larger transcripts, and in addition, that the intramolecular recombination of 2mu circle which occurs in yeast functions as a genetic switch to allow separate expression of two sets of genes on the 2mu circle genome.

Binding Sites↗

PubMed1992

Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma.

BACKGROUND: In atopic asthma, activated T helper lymphocytes are present in bronchial-biopsy specimens and bronchoalveolar-lavage (BAL) fluid, and their production of cytokines may be important in the pathogenesis of this disorder. Different patterns of cytokine release are characteristic of certain subgroups of T helper cells, termed TH1 and TH2, the former mediating delayed-type hypersensitivity and the latter mediating IgE synthesis and eosinophilia. The pattern of cytokine production in atopic asthma is unknown. METHODS: We assessed cells obtained by BAL in subjects with mild atopic asthma and in normal control subjects for the expression of messenger RNA (mRNA) for interleukin-2, 3, 4, and 5, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon gamma by in situ hybridization with 32P-labeled complementary RNA. Localization of mRNA to BAL T cells was assessed by simultaneous in situ hybridization and immunofluorescence and by in situ hybridization after immunomagnetic enrichment or depletion of T cells. RESULTS: As compared with the control subjects, the subjects with asthma had more BAL cells per 1000 cell that were positive for mRNA for interleukin-2 (P less than 0.05), 3 (P less than 0.01), 4 (P less than 0.001), and 5 (P less than 0.001) and GM-CSF (P less than 0.001). There was no significant difference between the two groups in the number of cells expressing mRNA for interferon gamma. In the subjects with asthma, mRNA for interleukin-4 and 5 was expressed predominantly by T lymphocytes. CONCLUSIONS: Atopic asthma is associated with activation in the bronchi of the interleukin-3, 4, and 5 and GM-CSF gene cluster, a pattern compatible with predominant activation of the TH2-like T-cell population.

Adult↗

PubMed1995

RNase H is responsible for the non-specific inhibition of in vitro translation by 2'-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers.

Ribonuclease H (RNase H) which recognizes and cleaves the RNA strand of mismatched RNA-DNA heteroduplexes can induce non-specific effects of antisense oligonucleotides. In a previous paper [Larrouy et al. (1992), Gene, 121, 189-194], we demonstrated that ODN1, a phosphodiester 15mer targeted to the AUG initiation region of alpha-globin mRNA, inhibited non-specifically beta-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of beta-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODN1, which limited RNase H activity on non-perfect hybrids. We report here that 2'-O-alkyl RNA-phosphodiester DNA sandwich analogues of ODN1, with the same phosphodiester window as MP-ODN2, are non-specific inhibitors of globin synthesis in wheat germ extract, whatever the substituent (methyl, allyl or butyl) on the 2'-OH. These sandwich oligomers induced the cleavage of non-target beta-globin RNA sites, similarly to the unmodified parent oligomer ODN1. This is likely due to the increased affinity of 2'-O-alkyl-ODN2 chimeric oligomers for both fully and partly complementary RNA, compared to MP-ODN2. In contrast, the fully modified 2'-O-methyl analogue of ODN1 was a very effective and highly specific antisense sequence. This was ascribed to its inability (i) to induce RNA cleavage by RNase H and (ii) to physically prevent the elongation of the polypeptide chain.

Alkylation↗

PubMed1985

Fluorescence microscopical hybridocytochemistry.

A new method has been developed to detect RNA-DNA hybrids in situ by fluorescence microscopy. This overcomes some of the disadvantages of autoradiographical detection of in situ hybridization, notably the low resolution and long exposure times needed. A procedure to label RNA at its 3'-terminus with a fluorochrome molecule has been developed. The optimal conditions for the cytochemical hybridisation reaction of this fluorochrome-labeled RNA were investigated using a model consisting of Sepharose beads to which nucleic acids has been bound. With RNA labeled both with 3H and rhodamine the hybridization reaction could be studied both biochemically by scintillation counting and cytochemically by microfluorometry. The fluorochrome-RNA bond was found to be unstable at higher temperatures. Therefore, the hybridization reaction had to be performed at room temperature in formamide-containing buffers. With fluorochrome-labeled complementary RNA kinetoplast DNA in Crithidia luciliae, adenovirus-5 DNA in infected KB cells and 5S rRNA, tRNA and cloned histone genes in polytene chromosomes of Drosophila could be localised. An immunocytochemical amplification method was developed that increased the sensitivity of the direct method. Several recently developed hybridocytochemical methods based on a combination of a hybridisation reaction in the first layer followed by immunocytochemical second layers, are described. The prospects of the application of the hybridocytochemical techniques in biomedical research, such as gene localisation and virus diagnosis, are discussed.

Autoradiography↗

PubMed1990

Antisense RNA inhibition of HPRT synthesis.

The abundant production of antisense hypoxanthine phosphoribosyltransferase (HPRT) RNA in NIH-3T3, COS, or HeLa cells leads to an inhibition of HPRT synthesis. HPRT enzyme levels in cells transfected with mouse HPRT antisense RNA expression vectors are reduced to less than 1% of parental cell activity, resulting in resistance to 6-thioguanine (6TG). The expression of antisense HPRT RNA leads to a marked reduction in the steady-state levels of endogenous HPRT mRNA. Furthermore, we demonstrate that intron-specific antisense RNA, complementary to sequences adjacent to splice donor or acceptor sites of the first intron of the mouse HPRT gene, are effective in depressing endogenous HPRT levels. These studies suggest that antisense RNA can inhibit gene expression in the nucleus, possibly by perturbing nuclear RNA processing.

Animals↗

PubMed1988

Cloning the human lysozyme cDNA: inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells.

Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has an 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a greater than 1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression.

Amino Acid Sequence↗

PubMed1980

Nuclear processing of viral high-molecular-weight RNA in cells infected with herpes simplex virus type 1.

Nuclear RNA isolated from cells infected by herpes simplex virus type 1, strain F, was fractionated on formamide-sucrose gradients into two major classes, greater and less than 45S. These two classes of labeled nuclear RNA were hybridized to viral DNA fragments generated by digestion with the restriction enzymes HindIII and BglII. Early in infection, only a few DNA fragments hybridized to RNA, with slight differences between the two classes. Late in infection, all DNA fragments hybridized, showing that all viral RNA was present in large precursor molecules greater than 14 kilobases. The fragments that correspond to late gene products hybridized more of the small RNA than the large RNA. This suggests that the mRNA corresponding to late genes accumulated after the large precursors have been cleaved. Large (greater than or equal to 45S) and small (< 45S) nuclear RNA and cytoplasmic RNA from cells late in infection were hybridized in excess to in vitro-labeled HindIII M and L fragments. More than 50% of the HIndIII M fragment annealed with the large nuclear RNA, but only 36% of it annealed with the cytoplasmic RNA. The HindIII L fragment hybridized large nuclear RNA and cytoplasmic RNA to the same extent (30% and 26%). These results suggest that RNA complementary to the HindIII M fragment, which is the template for immediate early polypeptides, was regulated in the nuclei at the posttranscriptional level. This seems to suggest that temporal regulation of RNA cleavage occurs in the nucleus.

DNA, Viral↗

PubMed1997

Corticotropin-releasing factor up-regulates its own receptor gene expression in corticotropic adenoma cells in vitro.

To investigate the expression of CRF receptor (CRF-R) in human corticotropic adenoma (hCA) cells, we analyzed messenger RNA (mRNA) levels of type-1 CRF-R (CRF-R1). Adenomas were obtained from 10 patients with Cushing's disease. Northern blot analysis using a rat CRF-R1 complementary RNA probe revealed a main hybridization band of 2.7 kilobases in all the hCAs. The CRF-R1 mRNA level significantly increased after 1 h, reached 15-fold the basal level at 8 h, and remained elevated 24 h after the addition of 10 nmol/L CRF in vitro. Dose dependency of the stimulatory effect of CRF was also demonstrated in hCA cells, whereas CRF down-regulated CRF-R1 mRNA levels in rat anterior pituitary (AP) cells. Treatment with dexamethasone or vasopressin decreased the CRF-R1 mRNA level in hCA cells, as observed in rat AP cells. In conclusion, we detected CRF-R1 mRNA in all hCAs tested. The CRF-R1 mRNA level was up-regulated by CRF itself in cultured hCA cells, in contrast to the down-regulation in rat AP cells.

Adenoma↗

PubMed1990

Elastin gene expression in elastotic human breast cancers and epithelial cell lines.

Elastosis is a prominent feature of the desmoplastic reaction in many invasive breast cancers. It is widely held that the elastic tissue is produced by fibroblastic cells of the breast stroma, but several studies have suggested that it derives from breast cancer epithelium. In studies directed to examining the mechanisms regulating desmoplasia in breast cancers, cell lines of human breast cancer derivation have been shown to synthesize immunoreactive tropoelastin in cell culture. Stromal fibroblasts, grown out from breast cancers, produced as much elastin as did nuchal ligament fibroblasts at similar passages. The human breast cancer cell lines, grown under similar conditions, produced elastin in culture at rates equivalent to 1.6-15% of those of the control fibroblastic cells. These included two estrogen receptor positive and one estrogen receptor negative cell types. Northern blot analysis of total RNA showed the presence, under high stringency conditions, of a 3.5-kilobase elastin mRNA band in both the fibroblastic cells and the cancer cell lines. In situ hybridization, with an elastin complementary RNA probe (prepared from a short segment of the translated region of human elastin mRNA), has been carried out on a selection of 21 invasive ductal breast cancers and 9 normal breast samples. It has been found that, while fibroblastic cells of the stroma and of the periductal region are responsible for elastin synthesis in most breast cancers, the malignant epithelium is a source of the elastin in the desmoplastic tissue of a significant proportion of such neoplasms. Vascular endothelium also expresses the elastin gene in some breast cancers. The elastotic elastin may have different cellular origins in different portions of a single ductal breast cancer. The results indicate that elastosis in breast cancers is very likely to be a complex process with multifactorial regulatory mechanisms. Subclassifying cancers according to the cellular source of the desmoplastic elastin, on the basis of in situ hybridization of elastin mRNA, may provide insights into the prognostic significance of elastosis in breast cancers.

Adult↗

PubMed2004

Synthesis and properties of fluorescent-labeled oligonucleotides containing amide-linked dinucleoside with pyrene.

Oligonucleotides containing 2'-pyrene modified uridine (Upy) exhibit the increase of the pyrene monomer emission when hybridize with the complementary RNA. In order to examine the effect of restricted C3'-endoconformation of the Upy residue for the enhancement of the pyrene monomer emission, an oligodeoxyribonucleotide containing amide-linked dinucleoside with a pyrene group at the sugar residue (UapyODN) was synthesized by use of 3'-carboxymethyl-5'-O-dimethoxytrityl-2'-O-(1-pyrenylmethyl)-3'-deoxyuridine. NMR analysis indicated that the furanose ring of the uridine derivative was fixed to C3'-endo conformation. The UapyODN showed increase of fluorescence intensity to 7-fold at 375 nm when hybridized with complementary oligoribonucleotide. This result suggests that the UapyODN can detect the target RNA.

Amides↗