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PubMed1984

The sequences of the N protein gene and intergenic region of the S RNA of pichinde arenavirus.

Two overlapping DNA clones representing more than half of the Pichinde arenavirus S RNA segment were cloned into pBR322 and their nucleotide sequences were determined. The analyses predict that the viral nucleocapsid protein (N) is encoded in a reading frame in the viral complementary RNA sequence starting at viral S RNA nucleotide residue 84 from the 3' end and terminating with an opal codon at residues 1767-1769. The position of the termination codon has been confirmed by primer directed dideoxynucleotide sequencing. The N protein has a calculated size of 62,911 Da and a net positive charge of +9. Viral complementary 15 S mRNA that directs the synthesis of N protein and hybridizes to the predicted N gene DNA has been identified in infected cell extracts. A second nonoverlapping reading frame in the viral complementary sequence originates at nucleotide position 1827 and remains open for at least 71 amino acids (i.e., the extent of the second clone). A long stretch of hydrophobic amino acids is near the amino terminus of this predicted gene product. Between the two reading frames is a 60-nucleotide-long noncoding intergenic region. This nucleotide sequence can be arranged in hairpin configuration involving 14 G-C and 4 A-U base pairs. The possible function of this intergenic region in the regulation of transcription and/or translation is discussed.

Amino Acid Sequence↗

PubMed1999

Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure.

Efficient gene control by antisense RNA requires rapid bi-molecular interaction with a cognate target RNA. A comparative analysis revealed that a YUNR motif (Y=pyrimidine, R=purine) is ubiquitous in RNA recognition loops in antisense RNA-regulated gene systems. The (Y)UNR sequence motif specifies two intraloop hydrogen bonds forming U-turn structures in many anticodon-loops and all T-loops of tRNAs, the hammerhead ribozyme and in other conserved RNA loops. This structure creates a sharp bend in the RNA phosphate-backbone and presents the following three to four bases in a solvent-exposed, stacked configuration providing a scaffold for rapid interaction with complementary RNA. Sok antisense RNA from plasmid R1 inhibits translation of the hok mRNA by preventing ribosome entry at the mok Shine & Dalgarno element. The 5' single-stranded region of Sok-RNA recognizes a loop in the hok mRNA. We show here, that the initial pairing between Sok antisense RNA and its target in hok mRNA occurs with an observed second-order rate-constant of 2 x 10(6) M(-1) s(-1). Mutations that eliminate the YUNR motif in the target loop of hok mRNA resulted in reduced antisense RNA pairing kinetics, whereas mutations maintaining the YUNR motif were silent. In addition, RNA phosphate-backbone accessibility probing by ethylnitrosourea was consistent with a U-turn structure formation promoted by the YUNR motif. Since the YUNR U-turn motif is present in the recognition units of many antisense/target pairs, the motif is likely to be a generally employed enhancer of RNA pairing rates. This suggestion is consistent with the re-interpretation of the mutational analyses of several antisense control systems including RNAI/RNAII of ColE1, CopA/CopT of R1 and RNA-IN/RNA-OUT of IS10.

Bacterial Proteins↗

PubMed1982

Papovavirus-related RNA sequences in human neurogenic tumours.

Thirty-two human brain tumours were investigated for the presence of papovavirus-related RNA sequences. Radiolabelled viral DNAs isolated from SV40, BKV, HDV, and BPV were hybridized in-situ on to tumour kryostat sections under conditions that detect complementary RNA. SV40-related RNA was detectable in 34% of all tumours tested but was not found in normal human brain tissue. SV40-positive tumours included 1/1 angiofibroma, 1/4 astrocytomas, 2/4 metastatic brain tumours, 1/1 melanoma, 3/7 meningiomas, 2/4 neurinomas, 1/2 oligodendrogliomas. Bovine papilloma virus-, BKV-, and HDV-related RNAs were absent from all tissues tested. The presence of SV40-related nucleic acids in a large number of human neurogenic tumours may reflect a viral involvement in the etiology of these tumours.

Astrocytoma↗

PubMed1980

Modification of cytidines in a Q beta replicase template: analysis of conformation and localization of lethal nucleotide substitutions.

The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in vitro by Q beta replicase, was investigated with sodium bisulfite, a reagent that selectively converts single-stranded cytidines to uridines. The reactivity of 45 of the 76 cytidines in MDV-1( + ) RNA was determined by nucleotide sequence analysis. Only 14 of these 45 cytidines were converted to uridine. Treatment of the RNA with methoxyamine, another single-strand-specific cytidine modification reagent, gave results in good agreement with the bisulfite data. The limited reactivity of MDV-1 ( + ) RNA with these reagents indicates that it is a highly structured molecule. A secondary structure consistent with the chemical modification data is proposed. Modification of MDV-1 ( + ) RNA by bisulfite renders it inactive as a template for RNA replication. This inactivation and the modification of the cytidines at the 3' end of the molecule occur at very similar rates. By using a short complementary RNA "mask" to protect just these cytidines, we demonstrated that the loss of activity resulted from their modification. This implies that one or more of the cytidines in the 3'-terminal sequence is required for template activity and that changes within this sequence can have lethal consequences. The effects of modification elsewhere in the sequence are discussed.

Base Sequence↗

PubMed2001

Conditioned activity to amphetamine in transgenic mice expressing an antisense RNA against the glucocorticoid receptor.

Glucocorticoids enhance the locomotion-stimulating and the rewarding properties of stimulant drugs. Amphetamine-induced conditioned activity was investigated in B6C3F1 (controls) and antisense transgenic mice. The latter expresses a neurofilament-promotor-driven antisense RNA complementary to a fragment of cDNA that codes for the mouse glucocorticoid receptor. This gene expression leads to approximately a 50% reduction in glucocorticoid receptor mRNA in the brain. Transgenic mice showed an increased novelty response when tested in an open field, in terms of both distance traveled and number of rearings. Moreover, they displayed enhanced amphetamine-induced conditioned activity. Behavioral sensitization was observed in controls, whereas behavioral tolerance developed in transgenic mice. These data support the concept of an enhanced stress response in these transgenic mice, rather than a general downregulation of the stress response because of impaired glucocorticoid receptor function.

Amphetamine↗

PubMed1998

Viral escape from antisense RNA.

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.

Antiviral Agents↗

PubMed2000

Antisense RNA sequences targeting the 5' leader packaging signal region of human immunodeficiency virus type-1 inhibits viral replication at post-transcriptional stages of the life cycle.

Antisense RNA has proven a potent inhibitor of gene expression and has the potential to inhibit retroviral replication at a number of stages in the virus life cycle by targeting both viral and cellular RNA sequences. Antisense RNA complementary to three target regions in the 5' leader/LTR of human immunodeficiency virus type-1 (HIV-1), the TAR region, the primer binding site and the splice donor (SD)-packaging signal (psi) region were stably expressed from the CMV IE promoter in Jurkat cells, and expression confirmed by RT-PCR. When challenged with HIV-1, cell lines expressing antisense RNA targeting the SD/psi region showed significant inhibition of replication (at up to 10(6) TCID 50/ml). These sequences were also expressed in lymphocytes after transduction using recombinant retroviruses and one sequence complementary to the SD/psi region inhibited replication of HIV-1. A co-transfection assay using COS-1 cells was also developed both to confirm the antiviral potential of these sequences, and to determine the predominant site of action of these molecules. Antisense RNAs targeting the psi region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore, analyses of relative levels of cellular and virion RNA from these assays suggest each of these antisense molecules exerts its effect at an early stage in the transcription-translation pathway, while the longer of the sequences also inhibited packaging of virion RNA. These results suggest that the packaging signal (psi) of HIV-1 represents an attractive target for antisense RNA-based gene therapy, although the main mode of action of such molecules may well be through antisense effects at an earlier stage of replication than packaging.

3T3 Cells↗

PubMed1985

Properties of rat and mouse beta-glucuronidase mRNA and cDNA, including evidence for sequence polymorphism and genetic regulation of mRNA levels.

cDNA clones containing partial sequences for beta-glucuronidase (beta G) were constructed from rat preputial gland RNA and identified by their ability to selectively hybridize beta G mRNA. One such rat clone was used to isolate several cross-hybridizing clones from a mouse-cDNA library prepared from kidney RNA from androgen-treated animals. Together, the set of mouse clones spans about 2.0 kb of the 2.6-kb beta G mRNA. Using these cDNA clones as probes, a genomic polymorphism for DNA restriction fragment size was found that proved to be genetically linked to the beta G gene complex. A fragment of beta G cDNA was subcloned into a vector carrying an SP6 polymerase promoter to provide a template for the in vitro synthesis of single-stranded RNA complementary to beta G mRNA. This provided an extremely sensitive probe for the assay of beta G mRNA sequences. Using either nick-translated cDNA or transcribed RNA as a hybridization probe, we found that mouse beta G RNA levels are strongly induced by testosterone, and that induction by testosterone is pituitary-dependent. During the lag period preceding induction, during the induction period itself, and during deinduction following removal of testosterone, beta G mRNA levels paralleled rates of beta G synthesis previously measured by in vivo pulse-labelling experiments. Genetic variation in the extent of induction affected either the level of beta G mRNA or its efficiency of translation depending on the strain of mice tested.

Amino Acid Sequence↗

PubMed2001

Impaired nephrogenesis in rats with congenital obstructive uropathy.

PURPOSE: Alterations caused by renal obstruction in developing kidneys are of particular interest in basic research of congenital obstructive uropathy. In rats nephrogenesis mainly occurs 7 to 10 days postnatally. Therefore, surgically induced neonatal ureteral obstruction in rats has been suggested to be analogous to congenital obstruction in the fetus. An attempt less prone to surgical artifacts and assessing even earlier developmental stages is to monitor the development of obstructed kidneys in rats with congenital obstructive uropathy. MATERIALS AND METHODS: Rats from an inbred strain with congenital renal obstruction in 70% of their littermates were observed. Morphologically, significant hydronephrosis was not detected before day 5 post partum and progressed with age. Unilateral obstructed kidneys were compared with contralateral kidneys and kidneys from healthy control animals at ages of 1, 5, 10, 18 and 32 days. A total of 72 renal units were investigated. The renal messenger RNA expression of renin and transforming growth factor-beta1 (TGF-beta1) was quantified by competitive quantitative reverse transcription polymerase chain reaction using a gene specific complementary RNA standard. RESULTS: In controls the gene expression of renin decreased from day 1 to day 18 and remained stable. TGF-beta1 expression increased during the first 10 days and then decreased again. Renin expression of the obstructed kidneys was reduced (p <0.05) on day 1, increased to a maximum versus controls (p <0.01) on day 10 and decreased to an unchanged elevated level (p <0.01) on days 18 and 32. Renin expression of the contralateral kidneys showed no significant alterations to control kidneys. Messenger RNA expression of TGF-beta1 of obstructed kidneys stayed decreased during the first 10 days (p <0.05), then increased excessively on day 18 (p <0.01) and slightly decreased on day 32. TGF-beta1 expression of the contralateral kidneys was parallel to controls on a slightly elevated level, increased on day 18 and returned to control level on day 32. CONCLUSIONS: Within the postpartum period of nephrogenesis gene expression of renin and TGF-beta1 was decreased in obstructed kidneys compared to controls. As the renin angiotensin system and TGF-beta1 have important functions in normal kidney development, these results suggest impaired nephrogenesis of congenital obstructed kidneys even before the onset of morphological signs of hydronephrosis. These features differ from surgical induced unilateral ureteral obstruction at birth and promise new insights into the pathophysiology of congenital obstructive uropathy.

Animals↗

PubMed1981

The construction, identification and characterisation of plasmids containing human alpha-lactalbumin cDNA sequences.

We describe the cloning of double-stranded cDNA synthesized from lactating human mammary gland total poly(A)-containing RNA, into the EcoRI site of the plasmid pAT153. Nine recombinants were shown to contain alpha-lactalbumin cDNA sequences as determined by positive hybridisation translation of complementary RNA. Restriction enzyme maps were determined for six of these. Alignment of the restriction map with the known amino acid sequence of human alpha-lactalbumin provided evidence that two plasmids, designated phO-53 and phB-35, contained the complete coding sequence of the primary translation product (pre-alpha-lactalbumin). Hybridisation studies using purified human, monkey and guinea-pig alpha-lactalbumin cDNA demonstrated that greater nucleotide sequence divergence has occurred within the rodents than the primates, and that rodent alpha-lactalbumin mRNAs retain regions of homology with primate alpha-lactalbumin mRNAs.

Animals↗

PubMed2005

Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference.

Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene-DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. (1)H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene-RNA/RNA and the pyrene-DNA/DNA duplexes.

Circular Dichroism↗

PubMed1977

Size and distribution of polyadenylic acid sequences in Drosophila polytene DNA and RNA.

[3H]Poly(U) hybridizes very rapidly to polytene DNA from Drosophila hydei. When hybridization is performed at 30 degrees C in 2 X SSC to a large excess of DNA, 95% of the poly(U) becomes ribonuclease resistant. Also, complementary RNA transcribed in vitro from polytene DNA hybridizes to poly(U). 023--0.25% of the DNA is composed of (dA)-rich sequences and 0.23--0.31% of cRNA hybridizes to [3H]poly(U). The length of the (dA)-rich sequences on the DNA and cRNA is 40 nucleotides. The Tm values of these hybrids formed between DNA or cRNA-poly(U) is 45 degrees C. The poly(A) fragments from cytoplasmic RNA ranged from 80 to 170 nucleotides in lenght, and migrated in polyacrilamide gels as a broad peak. The average sizes of the poly(A) fragments from the poly(A)-containing RNA transcribed by nuclei isolated from salivary glands in vivo or in vitro were 40, 70, 170 and 70 nucleotides, respectively. Hybridization in situ of [3H]-poly(U) to chromosome squashes indicated that the (dA)-rich sequences are randomly distributed over the whole genome.

Animals↗

PubMed1992

Dugbe Nairovirus M RNA: nucleotide sequence and coding strategy.

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.

Amino Acid Sequence↗

PubMed1978

Structure of the genome of Moloney murine leukemia virus: a terminally redundant sequence.

The genome of the Moloney strain of murine leukemia virus (Mo-MuLV) has been analyzed by digestion with ribonuclease T1 and separation of the digestion products by two-dimensional gel electrophoresis. Thirty large oligonucleotides isolated from such a fingerprint have been characterized. One of these oligonucleotides (number 21) was found to be present in twice the molar yield of the rest. The 30 oligonucleotides were mapped on the genome by determining their yields in various size classes of 3' terminal fragments of Mo-MuLV RNA. The physical map obtained in this way suggested that oligonucletoide 21 was present very near the 3' end of the geome as well as in another location near or at the 5' end. The genome structure suggested by these results was confirmed by analyzing oligonucleotides in Mo-Mulv RNA complementary to strong stop DNA, which is shown to be a copy of the 5' terminal 134 nucleotides of the MoMuLV genome. Some of the oligonucleotides in the RNA protected from RNAase digestion by hybridization to this DNA, including oligonucleotide 21, were present near both the 3' and 5' ends. Comparison of these with the nucleotide sequence of strong stop DNA shows that there is a terminal redundancy of 49-60 nucleotides in the Mo-MuLV genome RNA.

Base Sequence↗

PubMed2005

Synthesis and properties of ENA oligonucleotides targeted to human telomerase RNA subunit.

Oligonucleotides uniformly modified with 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) units were synthesized using the phosphoramidite method on a hundred-milligram scale for the evaluation of thermodynamic and chemical properties. The properties of these ENA oligonucleotides with the sequences targeted to human telomerase RNA subunit (hTR) were compared with those of GRN163, which is an oligonucleotide modified with N3'-P5' thiophosphoramidates. The melting temperatures of the duplexes of ENA oligonucleotides with complementary RNA were higher than that of the duplex of GRN163. Moreover, ENA oligonucleotide ENA-13 was more highly stable than GRN163 under acidic conditions (pH 5.0). ENA-13, which contained contiguous guanine sequences, could not form a G-quadruplex, which formation is not feasible for binding to hTR as an antisense molecule. The above findings suggest that ENA oligonucleotides may be useful for antisense therapeutic applications.

Humans↗

PubMed1983

Complementary DNA-25S ribosomal RNA hybridization: an improved method for phylogenetic studies.

In a new combination of techniques for ribosomal RNA hybridization, complementary DNA is synthesized on 25S ribosomal RNA fragments generated by mild alkali treatment, by the enzymatic addition of polyadenylic acid tails, hybridization of these tails with oligo deoxyribosylthymine, and reverse transcription in the presence of tritiated TTP. Hybridization reactions are performed in solution. Heteroduplexes are collected on diethylaminoethylcellulose filter discs after treatment with S1 nuclease. The problems presented by secondary rRNA structure are avoided by denaturation before reverse transcription and before hybridization. The high percentage of duplex formation (78-87%), the low standard deviation of relative binding (averaging +/- 1.30% relative binding), and small differences in reciprocal hybridizations (1.71-5.18% relative binding), as well as the elimination of complications resulting from differences in the number of rRNA cistrons in nuclear DNA, make this method preferable to the membrane-filter technique commonly used in phylogenetic classifications based on the homology of large rRNAs.

DNA↗

PubMed1992

EGF-R antisense RNA blocks expression of the epidermal growth factor receptor and suppresses the transforming phenotype of a human carcinoma cell line.

We have used an antisense approach to investigate the role of overexpression of the normal human epidermal growth factor (EGF) receptor in the transformed phenotype of KB cells, which are a tumor derived human cell line. Initial experiments performed in vitro, showed that antisense RNA complementary to the entire coding region (AS-FL) or to parts of the EGF-R mRNA (AS-3', AS-5', and AS-K) effectively blocked translation of EGF-R mRNA. In addition, upon microinjection into KB cells, the in vitro synthesized antisense RNAs were able to inhibit transiently the synthesis of EGF-R. Inhibition was concentration-dependent, both in vitro and in cells, and the most effective constructs were those complementary to the entire coding region (AS-FL) or to the 3'-coding end of the mRNA (AS-3'). Transfection of the same EGF-R antisense RNA constructs into the human epidermoid carcinoma KB cell line gave rise to several clones stably expressing elevated levels of antisense RNA and resulting in low residual levels of EGF receptor. The most reduced clones exhibited a totally restored serum-dependent growth and were severely impaired in colony formation and growth in agar. In addition the severity of the phenotype was directly proportional to the residual amount of EGF-R expressed. We conclude that over-expression of normal EGF-R plays a direct primary role in the development of the transformed phenotype of this human cancer cell line.

Carcinoma, Squamous Cell↗

PubMed1988

The glucocorticoid dexamethasone inhibits synthesis of interferon by decreasing the level of its mRNA.

Human fibroblasts were induced to secrete interferon (IFN) by treatment with the double-stranded RNA poly(inosinic).poly(cytidylic) acid or by infection with Newcastle disease virus. Treatment with 0.1-1 microM dexamethasone reduced the amount of IFN secreted by approximately 40-70%, respectively. A similar decrease in secretion of human IFN-beta was detected in dexamethasone-treated murine C127 cells that carry an IFN expression vector. These cells transcribe constitutively human IFN-beta under the control of a viral thymidine kinase promotor. Secretion of murine IFN induced by double-stranded RNA was also reduced in dexamethasone-treated C127 cells. The amount of IFN-beta mRNA present in fibroblasts and C127 cells was measured by hybridization to complementary RNA. Treatment with dexamethasone markedly reduced the level of IFN-beta mRNA present in both cells. The time course of this decrease was measured in C127 cells; 50 and 80% loss of IFN mRNA was observed after approximately 7.5 and 12 h, respectively. Murine IFN mRNA was also decreased in dexamethasone-treated C127 cells induced with double-stranded RNA. However, the rate of transcription of human IFN mRNA measured by run-on assays in isolated nuclei of dexamethasone-treated C127 cells was found to be comparable to that of control untreated cells. The finding that dexamethasone reduces the level of IFN mRNA transcribed under the control of both its own promotor and an unrelated promotor, together with the observation that dexamethasone does not apparently alter the rate of transcription of this mRNA, suggest that glucocorticoids may regulate IFN production by decreasing the level of its mRNA.

Animals↗