Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA, Complementary”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 703 records · Page 39Linked to original sources
PubMed1987

Human leukocyte antigens (HLA) class I and class II on sperm cells studied at the serological, cellular, and genomic levels.

The expression of human leukocyte antigens (HLA) on highly purified human ejaculated sperm cells was studied using the sensitive enzyme-linked immunosorbent assay (ELISA) technique and a wide panel of monoclonal antibodies to class I and class II HLA. In addition, the stimulatory capacity of these cells was tested in mixed cultures of lymphocytes and spermatozoa, and the levels of RNA homologous to the HLA class I and class II genes were determined. The results obtained using the ELISA indicate that the class I and class II HLA serologically defined antigens are weakly expressed on the cell surface of the mature spermatozoa. Highly purified sperm cells consistently stimulated heterologous lymphocytes but not when HLA-DR compatibility was observed between stimulator and responder. The proliferative response of lymphocytes induced by sperm cells was lower than the response obtained in a lymphocyte-lymphocyte combination, though the kinetics of the response were similar in both cases. In addition, it was found that spermatozoa contained RNA species homologous to HLA class II DR beta and DQ beta genes sequences but not to HLA class I sequences. The levels of these RNA species were significantly reduced after interferon stimulation. Lymphocytes that served as positive control were found to contain RNA complementary to both HLA class I and class II genes.

Antibodies, Monoclonal↗
PubMed2002

Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages.

Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage kappa3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the kappa3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10(-7) and 10(-8), beyond the resistance conferred by the PER element alone (less than 10(-6)). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.

Amino Acid Sequence↗
PubMed1994

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates.

Oligonucleotide (2-aminoethyl)phosphonates in which the backbone consisted of isomerically pure, alternating (2-aminoethyl)-phosphonate and phosphodiester linkages have been prepared and characterized. One of these single isomer oligonucleotides (Rp) formed a more stable duplex with DNA or RNA than its corresponding natural counterpart. Hybrid stability was more pH-dependent, but less salt-dependent than a natural duplex. The specificity of hybridization was examined by hybridization of an oligonucleotide containing one (2-aminoethyl)phosphonate to oligonucleotides possessing mismatches in the region opposite to the aminoethyl group. In contrast to oligonucleotides containing (aminomethyl)-phosphonate linkages, oligonucleotide (2-aminoethyl)phosphonates were completely stable to hydrolysis in aqueous solution. These oligonucleotides were resistant to nuclease activity but did not induce RNase H mediated cleavage of a complementary RNA strand. Incubation in a serum-containing medium resulted in minimal degradation over 24 hours. Studies of cell uptake by flow cytometry and confocal microscopy demonstrated temperature dependent uptake and intracellular localization. (2-Aminoethyl)phosphonates represent a novel approach to the introduction of positive charges into the backbone of oligonucleotides.

Aminoethylphosphonic Acid↗
PubMed1997

Enhanced expression of endothelin-1 gene in resistance arteries in severe human essential hypertension.

BACKGROUND: Endothelins are potent vasoconstrictors, and may also act as mitogens and hypertrophic agents. Expression of a member of this family of peptides, endothelin-1, is enhanced in the endothelium of blood vessels of rats with severe forms of hypertension, even in the absence of elevated plasma endothelin levels. In some of these hypertensive models enhanced endothelin-1 gene expression may contribute to vascular hypertrophy of small arteries and to elevation of blood pressure. OBJECTIVE: To establish whether endothelin-1 may play a role in essential hypertension in humans, in whom plasma levels are known to be usually within normal limits, by examining the expression of the endothelin-1 gene in resistance-size arteries of normotensive subjects, and in humans with mild and severe hypertension. METHODS: Using in-situ hybridization, the abundance of endothelin-1 messenger RNA transcripts was evaluated in small arteries of subcutaneous gluteal fat obtained by biopsy in normotensive and hypertensive patients. RESULTS: Vessels from five normotensive subjects and four untreated mild essential hypertensive patients did not exhibit topographically localized specific labeling with the antisense human endothelin-1 probe. Biopsies from four untreated hypertensive patients with moderate-to-severe blood pressure elevation, in contrast, showed a heavy density of grains on endothelial cells of small arteries of gluteal subcutaneous fat, corresponding to hybridization of the antisense human endothelin-1 complementary RNA probe with endothelin-1 messenger RNA. CONCLUSION: Some patients with moderate-to-severe essential hypertension, similar to some experimental rat models with severe blood pressure elevation, exhibit enhanced endothelial expression of the endothelin-1 gene. This is the first demonstration that overexpression of the endothelin-1 gene may occur in the vascular wall in a small sample of this subset of hypertensive patients. This pathophysiologic phenomenon could play a role in blood pressure elevation and perhaps in the pathogenesis of vascular hypertrophy. Treatment with endothelin receptor antagonists may offer a novel therapy for these moderate-to-severe hypertensive patients.

Adipose Tissue↗
PubMed2003

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions.

Myotonic dystrophy (DM1) is caused by the expansion of a trinucleotide repeat (CTG) located in the 3'untranslated region of the myotonic dystrophy protein kinase gene, for which currently there is no effective treatment. The data available suggest that misregulation of RNA homeostasis may play a major role in DM1 muscle pathogenesis. This indicates that the specific targeting of the mutant DMPK transcripts is essential to raise the rationale basis for the development of a specific gene therapy for DM1. We have produced a retrovirus which expresses a 149-bp antisense RNA complementary to the (CUG)13 repeats and to the 110-bp region following the repeats sequence to increase the specificity. This construct was introduced into human DM1 myoblasts, resulting in a preferential decrease in mutant DMPK transcripts, and effective restoration of human DM1 myoblast functions such as myoblast fusion and the uptake of glucose. It was previously shown that delay of muscle differentiation and insulin resistance in DM1 are associated with misregulation of CUGBP1 protein levels. The analysis of CUGBP1 levels and activity in DM1 cells expressing the antisense RNA indicated a correction of CUGBP1 expression in infected DM1 cells. We therefore show that current antisense RNA delivered in vitro using a retrovirus is not only capable of inhibiting mutant DMPK transcripts, but also can ameliorate dystrophic muscle pathology at the cellular levels.

Blotting, Northern↗
PubMed1980

Studies with a cold-recombinant A/Victoria/3/75 (H3N2) virus. I. biologic, genetic, and biochemical characterization.

A cold-recombinant virus CR 22, was derived from an attenuated cold-adapted parent strain. A/Ann Arbor/6/60 (H2N2), and a wild-type parent strain, A/Victoria/3/75 (H3N2). Antigenic analysis showed that CR 22 possesses the hemagglutinin and neruaminidase surface antigens derived from the A/Victoria/3/75 (H3N2) parent. From studies of virus-induced polypeptides using polyacrylamide gel electrophoresis, it was deduced that a polymerase protein, P1, is coded and by an RNA segment derived from the wild-type parent; all other genetic elements are derived from the attenuated parent. The attenuated parent and the recombinant CR 22 both possess temperature-sensitive (ts) lesions, evident by restriction of multiplication in fertile chicken eggs or in Madin-Darby canine kidney cell cultures at greater than or equal to 38 C. Genetic analysis of CR 22 by complementation tests using Hong Kong and Wilson Smith neurotropic ts mutants gave evidence for a ts lesion in the genetic elements coding for complementary RNA.

Animals↗
PubMed1975

Human RNA transcripts in man-mouse somatic cell hybrids. II. Thermal denaturation studies and Cot analysis.

The present report confirms an earlier finding that human RNA transcripts from a man-mouse somatic cell hybrid that has regularly retained only the human X chromosome can be identified by molecular hybridization on nitrocellulose filters. From thermal denaturation studies it is concluded that molecular hybrids between hybrid cell and human nucleic acids have higher Tm's, and thus greater specificity, than those between mouse and human nucleic acids. Additional data, utilizing the kinetics of molecular hybridization in solution (Cot analysis), demonstrate the presence of "few gene copy" DNA sequences in the above-mentioned hybrid cells that are complementary to human (HeLa) HnRNA. Also shown is a fraction of hybrid cell HnRNA that is complementary to "few gene copy" DNA sequences of human DNA. Thus, the experiments reported may represent a specific assay for identifying reiterated and "few gene copy" DNA sequences of the human X chromosome and their complementary RNA. Theoretically this experimental approach may be extended to any chromosome. Some of the short-term and long-term perspectives of these types of studies are discussed.

Animals↗
PubMed1990

Sequence and transcriptional analysis of the barley ctDNA region upstream of psbD-psbC encoding trnK(UUU), rps16, trnQ(UUG), psbK, psbI, and trnS(GCU).

A 6.25 kbp barley plastid DNA region located between psbA and psbD-psbC were sequenced and RNAs produced from this DNA were analyzed. TrnK(UUU), rps16 and trnQ(UUG) were located upstream of psbA. These genes were transcribed from the same DNA strand as psbA and multiple RNAs hybridized to them. TrnK and rsp16 contained introns; a 504 amino acid open reading frame (ORF504) was located within the trnK intron. Between trnQ and psbD-psbC was a 2.24 kbp region encoding psbK, psbI and trnS(GCU). PsbK and psbI are encoded on the same DNA strand as psbD-psbC whereas trnS(GCU) is transcribed from the opposite strand. Two large RNAs accumulate in barley etioplasts which contain psbK, psbI, anti-sense trnS(GCU) and psbD-psbC sequences. Other RNAs encode psbK and psbI only, or psbK only. The divergent trnS(GCU) located upstream of psbD-psbC and a second divergent trnS(UGA) located downstream of psbD-psbC were both expressed. Furthermore, RNA complementary to psbK and psbI mRNA was detected, suggesting that transcription from divergent overlapping transcription units may modulate expression from this DNA region.

Amino Acid Sequence↗
PubMed1986

Inducible production of c-fos antisense RNA inhibits 3T3 cell proliferation.

Antisense RNA complementary to c-fos mRNA was produced in mouse 3T3 cells by gene transfer techniques. Transcriptional units were constructed consisting of a steroid-inducible mouse mammary tumor virus (MMTV) promoter, mouse or human 5' c-fos gene fragments in either the sense (5' to 3') or antisense (3' to 5') orientation, and splice and poly(A) signals from the human beta-globin gene. A gene that confers neomycin resistance was included in the vectors to allow isolation of stable transformants. Dexamethasone caused a marked induction of hybrid MMTV-fos-globin RNA. Induction of the hybrid transcript containing antisense c-fos RNA decreased colony formation following DNA transfer and inhibited the proliferation of cells into which the antisense transcriptional unit had been integrated. In contrast, colony formation and cell proliferation were not inhibited by induction of hybrid RNA containing c-fos RNA sequences in the sense orientation. These results indicate that the strategy of generating antisense RNA to inhibit gene expression may be useful in delineating the function of protooncogenes. The c-fos gene product appears to have a required role in normal cell division.

Animals↗
PubMed1976

Analysis of in vitro transcription products of intracellular vesicular stomatitis virus RNA polymerase.

The intracellular transcriptase complex of vesicular stomatitis virus-infected L cells synthesized RNA complementary to the entire infectious virus genome at either 37 degrees C or 28 degrees C in vitro. Not all sequences were present at the same frequency, however; copies of that segment of the genome common to the LT defective particles were present at 20 to 100 times higher frequently than copies of the genome segment common to the ST defective particle. The less frequent region was transcribed somewhat more effectively at 28 degrees C than at 37 degrees C. The results suggest that transcriptional regulation rather than selective degradation is responsible for the differential accumulation of RNA.

Base Sequence↗
PubMed2003

Synthesis, thermal stability and resistance to enzymatic hydrolysis of the oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridines.

The synthesis of oligonucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridine (D) is described, and thermal stability and resistance to enzymatic hydrolysis of the ODNs are compared with ODNs containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine (H). The ODNs containing D and the complementary RNA demonstrated a duplex thermal stabilization of 0.4-3.9 degrees C per modification depending on the position and the number, while the ODNs containing H with the RNA showed slightly less effective thermal stabilization. Further more, the ODNs containing D were found to be more resistant to nucleolytic hydrolysis, not only by snake venom phosphodiesterase (SVPD; a 3'-exonuclease) but also by DNase I (an endonuclease). The half-life of the 17mer containing five molecules of D against nucleolytic hydrolysis by SVPD was 240 times greater than the unmodified 17mer ODN, which is 1.8 times greater than the ODN containing 5Hs in the same sequence. Against DNase I, the same ODN containing 5Ds was 24 times greater stable than the unmodified 17mer and 15 times more stable than the ODN containing 5Hs. We also examined whether the duplexes formed by the ODNs containing D and the complementary RNAs could be a substrate of Escherichia coli RNase H. It was revealed that a minimum of five contiguous unmodified 2'-deoxyribonucleosides between Ds was required to constitute a substrate of E.coli RNase H. Thus, the ODN with Ds and at least five contiguous unmodified 2'-deoxyribonucleosides between Ds was found to be a candidate for a novel antisense molecule.

Base Sequence↗
PubMed2003

In vitro analysis of RNA interference in Drosophila melanogaster.

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.

Animals↗
PubMed1998

Domain structure and RNA annealing activity of the Escherichia coli regulatory protein StpA.

The Escherichia coli regulatory protein StpA bears striking similarity to the chromatin-associated protein H-NS. These two proteins have many structural, functional and mechanistic parallels. Although H-NS is more abundant in the cell, both proteins act as transcriptional regulators, both bind to curved DNA and both restrain DNA supercoils. However, StpA is better able to promote RNA annealing and trans-splicing in vitro. In this study, phylogenetic analyses and experiments to examine the protease sensitivity of StpA and H-NS suggest a similar structure for the two proteins. Both proteins consist of two structured domains separated by an exposed protease-sensitive linker. The N-terminal (StpA-NterL) and C-terminal (StpA-CterL) domains of StpA, as well as the full-length StpA and H-NS proteins, were cloned, overproduced in E. coli and purified to homogeneity. StpA-CterL, but not StpA-NterL, promotes strand annealing of complementary RNA oligonucleotides and in vitro trans-splicing of a model group I intron. Both StpA and StpA-CterL exhibited stronger RNA-modulating activity than H-NS. Phylogenetic analyses showed that the N-terminal and C-terminal domains can exist autonomously. The phylogenetic and experimental data are compatible with a two-domain model for StpA and H-NS, with independently functioning modules joined by a non-conserved linker, and with the observed RNA-related activities residing entirely within the C-terminal domain.

Amino Acid Sequence↗
PubMed1996

Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets.

Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.

Amides↗
PubMed1991

Cloning of non-polyadenylated RNAs from rat brain.

Rodent brain has been reported to contain a fraction of non-polyadenylated (poly(A)-) mRNA that includes about 100,000 different sequences, most of which are not found in the poly(A)+ fraction. We have prepared a cDNA library of low-abundance poly(A)- RNAs from rat brain polysomes, and have characterized three clones in detail. Two of the clones hybridize on Northern blots to poly(A)+ RNAs from brain. Dot blot hybridization and RNase protection assays demonstrate that although the bulk of the RNA complementary to these clones is present in the poly(A)- fraction, a small portion (7-21%) is present in the poly(A)+ fraction. Our results suggest that the poly(A)-mRNA fraction from rat brain may not contain sequences that are different from those in the poly(A)+ fraction.

Animals↗
PubMed1990

Endonucleolytic cleavage of a maternal homeo box mRNA in Xenopus oocytes.

We have identified a messenger RNA (mRNA) sequence from a Xenopus homeo box-containing gene that is the target for a sequence-specific endoribonuclease in vivo. Synthetic RNA transcribed from an allele of the maternal gene Xlhbox2B is efficiently cleaved when injected into Xenopus oocytes. The cleavage sequence lies between the protein-coding region and a 600-base 3'-untranslated region. Intermediates in degradation are readily observed: Both the 5' and 3' products of cleavage are recovered, thus showing that the cleavage activity is an endonuclease. When a 90-base region of the Xlhbox2B sequence is inserted into a second homeo box RNA that is normally stable, it is sufficient to confer an identical cleavage reaction on the hybrid RNA. The cleaved region contains a repeated sequence motif and is cut at multiple sites. Inhibition of translation does not affect the rate or extent of cleavage, while the coinjection of antisense RNA complementary to the 90-base region completely blocks the reaction. Because most mRNAs are not found on polysomes during oogenesis, translation-independent cleavage at such sites may provide a novel post-transcriptional mechanism to regulate the amount of mRNA available for embryogenesis.

Alleles↗
PubMed1986

Formation of lariats and circles in self-splicing of the precursor to the large ribosomal RNA of yeast mitochondria.

Self-splicing of the precursor to large ribosomal RNA of yeast mitochondria leads not only to circles but also to lariats, structures that have not been observed before as products of self-splicing. Lariats were studied by electron microscopy after hybridization with an RNA complementary to the 3' half of the precursor. This leads to differentiation in at least two classes of lariats that vary in the position of the branch point. In all lariats the tail carries the 3' end, which suggests that a 5' end is used for branch formation with an internal nucleotide. The circles are formed from excised introns. They lack only three nucleotides encoded by mitochondrial DNA along with the 5'-terminal G added in the course of self-splicing. The diverse number of self-splicing products arising in vitro testifies to the considerable reactivity of this intron. The formation of lariats in an RNA catalyzed reaction may have implications for views on the mechanism of splicing of nuclear pre-mRNAs.

Catalysis↗
PubMed1997

The human gene for the regulatory subunit RI alpha of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase: two distinct promoters provide differential regulation of alternately spliced messenger ribonucleic acids.

The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.

Aged↗