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 1,549 records · Page 86Linked to original sources
PubMed1995

A vector for the synthesis of cRNAs encoding Myc epitope-tagged proteins in Xenopus laevis oocytes.

We describe a plasmid, pNKS2-myc, designed for convenient in-frame fusion of an antibody-specific epitope sequence to the N terminus of a desired cDNA and subsequent synthesis of transcripts that direct the synthesis of the tagged polypeptide in Xenopus laevis (Xl) oocytes. pNKS2-myc contains an SP6 promoter, followed by the translation initiation sequence of the Na,K-pump beta 3 subunit of Xl and the sequence encoding an epitope derived from the human c-myc proto-oncogene product. Appropriate restriction sites allow one to insert virtually any desired cDNA fragment directly behind the epitope-specific sequence and before a long poly(A) tail. After linearization with EcoRI or NotI, polyadenylated cRNA can be synthesized that is efficiently translated in Xl oocytes. The utility of pNKS2-myc is demonstrated by cloning cDNAs coding for Na,K-pump subunits into this vector and injecting the corresponding cRNAs into oocytes. The tagged mouse beta 1 and beta 2 subunit isoforms could be purified from detergent extracts of these cells by immunoprecipitation with a generally available monoclonal antibody (mAb) to the tag, 9E10, as well as with specific mAb that recognize individual beta subunit isoforms. Under native conditions, endogenous and coexpressed exogenous alpha 1 subunits (the catalytic subunit of the Na,K-pump) were co-precipitated, indicating that the N-terminal addition of the decapeptide epitope has no adverse effect on the folding of beta subunits nor on their assembly with alpha subunits. Furthermore, the Myc-specific mAb likewise precipitated a Myc-tagged Na,K-pump alpha 1 subunit together with any of the co-synthesized beta subunits.

Amino Acid Sequence↗
PubMed2002

Natural antisense (rTSalpha) RNA induces site-specific cleavage of thymidylate synthase mRNA.

The 3' untranslated region (UTR) of rTSalpha RNA is complementary (i.e., antisense) to human thymidylate synthase (TS) RNA. When HEp2 cells (human epidermoid carcinoma) progressed from late-log to plateau phase growth, ribonuclease protection assay (RPA) revealed an inverse correlation between the levels of rTSalpha RNA and TS mRNA, suggesting a possible effect of rTSalpha RNA on TS mRNA levels. HEp2 cells expressing a Tet-On transactivator were transiently co-transfected with pHook-1 and a construct containing rTSalpha (protein and antisense RNA), rTSalphaDelta3' (rTSalpha protein only), rTSalpha-3' (antisense RNA-luciferase) or luciferase. Transfected cells were selected and evaluated for the effects of induced transgene expression on TS mRNA. Induced expression of transfected rTSalpha or rTSalpha-3', but not rTSalphaDelta3' or luciferase, resulted in decreased TS mRNA levels as measured by RPA. These results demonstrated that the antisense region of rTSalpha RNA is necessary and sufficient for this down-regulation of TS mRNA. RPA for TS mRNA also showed the enhanced appearance of two partial-length protected fragments in rTSalpha or rTSalpha-3' transfected cells. RPA stringency evaluations and primer extension assays indicated that TS mRNA is cleaved in vivo in a site-specific manner. These data demonstrate that rTS gene expression likely plays a role in down-regulating TS through a natural RNA-based antisense mechanism.

3' Untranslated Regions↗
PubMed1997

Control of complexity constraints on combinatorial screening for preferred oligonucleotide hybridization sites on structured RNA.

We have explored the use of short (10-mer), fully sequence-randomized oligonucleotide libraries for affinity-based screening in solution for energetically preferred sites of hybridization of a model 47-nucleotide (nt) mutant Ha-ras mRNA stem-loop fragment. In characterizing the model, binding studies using either a gel mobility-shift assay or an RNase ONE footprinting assay indicated the presence of a greatly preferred hybridization site for individual antisense RNA oligonucleotides on the 5'-most side of the ras RNA 19-nt loop. However, initial attempts to affinity-titrate combinatorial uniform 2'-O-methyl-substituted oligonucleotide libraries for selective binding to this 5'-loop site using an RNase ONE footprinting assay that can discriminate between binding to different sites on ras RNA were unsuccessful. By reducing the complexity of the library to a mix of seven RNA oligonucleotides complementary to a range of sites on ras RNA and with no self-complements, footprinting evidence for binding was obtained but was characterized by ras RNA site-specific binding constants differing dramatically from binding constants for individual oligonucleotides. The library complexity was reduced further to three different cases of two RNA oligonucleotides, one of which for all cases was the highest affinity 5'-loop complement. Detailed kinetic and thermodynamic binding analyses revealed a good fit of the data to independent (5'-loop and ascending stem sites), competitive (overlapping 5'-loop sites), or mutually allosteric (5'-loop and 3'-loop sites) formalisms and an energetics description showed that ras 5'-loop site-specific binding could be achieved by affinity titration only for the independent case. Reconstruction of events with the full complexity library suggested that there was the emergence of multiple, linked binding interactions and implied that successful hybridization affinity screening would be achieved only if all possible bimolecular binding interactions of individual library oligonucleotides with target RNA could be made mutually independent. Accordingly, by holding the calculated concentration of unique oligonucleotide sequences of a full complexity DNA library well below the value for the dissociation constant for binding of individual complement to the 5'-loop site and then titrating the concentration of ras RNA through this value, hybridization specific to the 5'-side of the ras loop was demonstrated as assayed either by sequential gel mobility-shift resolution of bimolecular complexes and RNase ONE footprinting in situ in gel slices or by RNase H cleavage of complexes in solution. Because this strategy uses an unbiased oligonucleotide library it should combinatorially identify energetically preferred hybridization sites on folded RNA targets of any sequence and of undetermined structure. This should enable a focused in vitro optimization of antisense oligonucleotide length, sequence, and chemical composition for preferred site binding affinity and specificity which, in turn, may be expected to provide for enhanced biological potency and specificity (Lima et al., 1996). Finally, the complexity constraints encountered and the fundamental requirement to control them presented here also should be applicable to interactions with any biomolecule target of any chemical class of combinatorial library when screened in solution in pooled mixes.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

The detection of prostate cells by the reverse transcription-polymerase chain reaction in the circulation of patients undergoing transurethral resection of the prostate.

OBJECTIVE: To determine whether prostate cells are disseminated into the circulation of patients after transurethral resection of the prostate (TURP), as assessed by a reverse transcription-polymerase chain reaction (RT-PCR) assay for prostate specific antigen (PSA) mRNA. PATIENTS AND METHODS: Fifty-one patients, comprising 34 with benign prostatic hyperplasia (BPH) and 17 with prostate cancer who were undergoing routine TURP, had blood samples taken before and 30 min after surgery. The blood mononuclear cell layer was isolated by density-gradient centrifugation and total RNA extracted. Complementary DNA was synthesized by RT of the RNA. The target PSA sequence was amplified by PCR with specific PSA primers and the product detected on agarose-gel electrophoresis with ethidium bromide staining. RESULTS: Five patients (all with prostate cancer) were positive on PSA RT-PCR before surgery and remained positive after TURP. Of the remaining 46 patients, five (11%; three with BPH and two with cancer) changed from a negative to a positive result after TURP. CONCLUSION: In a proportion of patients TURP causes the dissemination of prostate cells into the circulation which are then detectable by RT-PCR of PSA mRNA.

Aged↗
PubMed1980

Synthesis in high yield of complementary DNA of retroviral RNA.

Mouse mammary tumor virus RNA was transcribed in vitro with avian myeloblastosis virus reverse transcriptase in the presence of calf thymus DNA oligomers. The yield of cDNA was markedly enchanced by increasing the concentration of the enzyme as well as primers in the reaction mixture. The average size of cDNA was approximately 200 residues, and it was not affected by the concentration of deoxynucleoside triphosphates when saturating concentration of enzyme was used. Nearly all of the viral sequences were represented in cDNA and it formed hybrids of high fidelity with viral RNA. Similar results were obtained when murine leukemia virus (AKR) RNA was transcribed. These observations will be useful for synthesizing cDNA of RNAs that are not easily available in sufficient quantities.

Avian Leukosis Virus↗
PubMed1981

Sequence of DNA complementary to a small RNA segment of influenza virus A/NT/60/68.

A small RNA segment from the influenza virus strain A/NT/60/68 (H3N2) was converted to cDNA and then to double-stranded DNA using synthetic oligodeoxynucleotide primers. The double-stranded form was cloned into the bacteriophage M1 3mp7. Clones yielding single-strand recombinant templates in opposite orientation were sequenced by the Sanger dideoxynucleotide chain termination technique. The small viral RNA was 422 nucleotides long and the evidence indicated that it was formed by internal deletion of segment 3. It also contained sequences homologous to segment 1.

Base Sequence↗
PubMed1992

RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur between replicating MHV RNAs and RNA fragments which do not replicate, suggesting the potential of RNA recombination for genetic engineering.

Base Sequence↗
PubMedDate not supplied

[The context analysis of polynucleotide sequences. II. Inverted repeats and complementary palindromes in RNA-polymerase genes].

A new approach to the reconstruction of the RNA secondary structure is suggested on the basis of the method of contextual analysis of polynucleotide sequences. The coding gene regions of beta-, beta'-, sigma-subunits of E. coli RNA polymerase and of phage T7 RNA polymerase were analysed. The clusters of non-random inverted repeats were found in all these genes. The mRNA coded by them can be folded into compact secondary structures. The latter are formed by quite long helices with a few cases of mispairing.

Chromosome Inversion↗
PubMed1975

Demonstration that a mouse immunoglobulin light chain messenger RNA hybridizes exclusively with unique DNA.

32P-labeled light chain messenger RNA was prepared from mouse MOPC 21 myeloma cells. The messenger RNA was hybridized to purified repetitive nuclear DNA and both the hybridized (repetitive 32P-RNA) and nonhybridized (nonrepetitive 32P-RNA) fractions were isolated. Only the nonhybridized RNA gave a T1 ribonuclease fingerprint showing oligonucleotides derived from the variable and constant regions of the light chain messenger RNA. In addition, this fingerprint showed oligonucleotides derived from the untranslated regions of the light chain messenger RNA. The nonrepetitive 32P-RNA was shown to rehybridize only with the unique fraction of total nuclear DNA. The rapidly hybridizing part of the unfractionated 32P-RNA preparation, therefore, is not a component of the light chain messenger RNA itself. Complementary DNA was prepared with reverse transcriptase using unlabeled light chain messenger RNA as template, and the transcripts were fractionated into various size classes. Complementary DNA molecules greater than 900 bases in length hybridized with both the initial messenger RNA and with the nonrepetitive 32P-RNA but failed to hybridize with excess purified repetitive 32P-RNA. The rapidly hybridizing component of the messenger RNA fraction, therefore, does not appear to be transcribed by reverse transcriptase. It is concluded that, under the experimental conditions used, the light chain messenger RNA hybridizes exclusively with unique DNA.

Cell Line↗
PubMed2003

Prioritized selection of oligodeoxyribonucleotide probes for efficient hybridization to RNA transcripts.

Only a small fraction of short oligonucleotide probes bind efficiently to complementary segments in long RNA transcripts. Technologies such as array-based transcript profiling and antisense control of gene expression would benefit greatly from a method for predicting probes that bind well to a given target RNA. To develop an algorithm for prioritizing selection of probes, we have analyzed predicted thermodynamic parameters for the binding of several large sets of probes to complementary RNA transcripts. The binding of five of these sets of probes to their RNA targets has been reported by others. In addition, we have used a method for light-directed synthesis of oligonucleotide arrays that we developed to generate two new arrays of surface-bound probes and measured the binding of these probes to their RNA targets. We considered predicted free energies for intramolecular base pairing of the oligonucleotide and its RNA target as well as the predicted free energy of intermolecular hybridization of probe and target. We find that a reliable predictor of probes that will hybridize significantly with their targeted transcripts is the predicted free energy of hybridization minus the predicted free energy for intramolecular folding of the probe.

Green Fluorescent Proteins↗