Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleoprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells.
...
PMID:RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection. 213 10

We have developed an assay that uses phenyl boronate agarose column chromatography to measure the capping efficiencies of RNA polymerases used for in vitro transcription of cloned cDNAs. Capped 32P-labeled ovalbumin mRNAs were synthesized by in vitro run-off transcription with SP6 or T7 RNA polymerase in the presence of cap analogs and digested to completion with T1 and T2 RNase. The resulting 3'-nucleoside monophosphates (NMPs) and cap structures were separated by chromatography on phenyl boronate agarose, and the ratio of radioactivity between the two was used to estimate the extent of transcript capping.
...
PMID:A simple assay for determining the capping efficiencies of RNA polymerases used for in vitro transcription. 226 28

A method for the normalization of multiple RNA samples is described. This method exploits the recently developed technology which allows the synthesis of single-stranded, specific RNA molecules in vitro using either SP6 or T7 RNA polymerase to prepare an external standard cRNA. When this external standard cRNA is added to cell samples at the time of lysis, it becomes a stable, integral part of the RNA content of each sample, which can easily and reproducibly be detected and quantitated by either Northern blot or RNase protection. The feasibility of this approach to normalization has been tested in a mouse 3T3 cell model system. In multiple samples, the relative levels of this externally added standard transcript are shown to closely parallel the relative levels of an internal standard control transcript as well as the amount of RNA determined by spectrophotometric analysis. The data obtained demonstrate that an externally added, in vitro-synthesized transcript can serve as an accurate, universal means of normalizing multiple RNA samples, since it is not dependent on sample RNA concentration, species, cell or tissue type, or experimental manipulation.
...
PMID:Normalization of multiple RNA samples using an in vitro-synthesized external standard cRNA. 244 10

Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF.
...
PMID:Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines. 245 52

We identified a Mg2+ dependent 5' exo-ribonuclease and an RNA ligase in cell-free extracts of Trypanosome brucei. The exo-ribonuclease in S100 or nuclear extracts, removes about 20 nts from the 5' end of SP6 derived capped as well as uncapped RNA and then stops. In contrast to the activity of the exo-ribonuclease on capped SP6 mini-exon transcripts, the exonuclease cannot degrade trypanosome-derived mini-exon transcripts or the mini-exon located at hsp 70 mRNAs. We therefore assume that the four secondary base modifications adjacent to the mini-exon cap, generated in vivo, confer resistance to the exo-ribonuclease. After exonuclease shortening of SP6 transcripts, an RNA ligase catalizes intramolecular ligation, generating a 3'-5' phosphodiester bond in a Mg2+ and ATP dependent reaction.
...
PMID:A 5' exo-ribonuclease and RNA ligase of T. brucei. 246 Aug 26

We used a ribonuclease cleavage assay to screen for insulin receptor mRNA sequence alterations in 12 patients with syndromes of severe insulin resistance. Uniformly labeled [32P]antisense RNA probes complementary to insulin receptor mRNA were prepared by an SP6 or T7 RNA polymerase transcription reaction. Four probes ranging in size from 670-1470 bases were used to examine the entire 4.2-kilobase receptor protein-coding region. Patient RNA samples were hybridized to individual probes in solution, and mismatched sequences were detected by susceptibility to cleavage by a mixture of RNAses A and T1. The method was validated with insulin receptor mRNAs from cells transfected with cDNA constructs bearing known point and deletion mutations. Alterations in the insulin receptor mRNA sequence of two patients were detected. A patient with the type A syndrome of severe insulin resistance (A2-Boston) had a mutation in the insulin receptor beta-subunit mRNA sequence that localized to the region coding for amino acid residues 1174-1211 near the tyrosine kinase domain. The second alteration was a sequence polymorphism in the insulin receptor alpha-subunit mRNA in a patient with lipoatropic diabetes (LA-2) that localized to a region within amino acids 268-272. Direct sequence analysis revealed that the ribonuclease cleavage sites in patients A2-Boston and LA-2 were due to distinct single base changes in the insulin receptor gene and mRNA. Additional insulin receptor mRNA sequence polymorphisms were also identified as mismatches between the labeled RNA probes used and mRNA from several cultured human cell types. This study demonstrates that ribonuclease cleavage can rapidly detect and localize insulin receptor mRNA sequence mutations and polymorphic variations as small as single base changes. Further analysis of insulin receptor mRNA sequence alterations identified in this way may elucidate a possible genetic basis for functional insulin receptor defects in patients with severe insulin resistance and can also reveal some insulin receptor sequence polymorphisms that occur in the population at large.
...
PMID:Insulin receptor messenger ribonucleic acid sequence alterations detected by ribonuclease cleavage in patients with syndromes of insulin resistance. 273 94

The interactions between the nucleocapsid protein N and either RNA or the phosphoprotein NS of vesicular stomatitis virus (VSV) were studied by the transcription of N and NS mRNAs from SP6 vectors, followed by translation in a rabbit reticulocyte lysate. Nascent N protein bound tightly to added labeled RNA, as well as to endogenous RNA in the reticulocyte lysate. This binding was demonstrated by three independent techniques. First, labeled N protein and labeled RNA migrated identically as a series of sharp, closely spaced bands in a nondenaturing gel system. Second, translated N protein behaved as a stable ribonucleoprotein complex in CsCl gradients and sedimented to the same density as the authentic N-RNA template of VSV. Third, translated N protein protected a series of labeled RNA fragments from digestion by RNase A. None of the three RNA-binding criteria was satisfied by either translated NS protein or two deletion mutants of N protein or by other components of the reticulocyte lysate. The evidence suggests that the observed binding of RNA by nascent N was not RNA sequence specific, in contrast to the encapsidation process during VSV replication. Moreover, the prior formation of N-NS complexes totally abolished the observed binding of RNA by N. Thus, we propose that NS may be responsible for conferring the sequence specificity of the RNA binding that occurs during VSV genome replication.
...
PMID:Complex formation with vesicular stomatitis virus phosphoprotein NS prevents binding of nucleocapsid protein N to nonspecific RNA. 283 93

A solution hybridisation assay has been developed which allows the quantitation of specific viral (RNA) sequences in infected cells. The assay makes use of single-stranded (ss) RNA probes of known polarity synthesised at high specific activity in vitro from cDNA clones of the relevant viral gene by the SP6 or T7 RNA polymerase. These probes are used together with samples containing the RNA to be detected at a known concentration to construct a calibration curve to relate RNase resistant radioactivity following solution hybridisation to amount of RNA. The amount of RNA in experimental samples is then determined using the calibration curve that is produced each time the assay is performed. The UKtc strain of Rotavirus growing in BSC-1 cells was used to develop this method but with the substitution of suitable cDNA clones it could be applied to any viral system.
...
PMID:A rapid and sensitive solution hybridisation assay for the quantitative determination of specific viral RNA sequences. 285 3

A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.
...
PMID:Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA. 288 93

The ribonucleoprotein (RNP) structures of the pre-mRNA and RNA processing products generated during in vitro splicing of an SP6/beta-globin pre-mRNA were characterized by sucrose gradient sedimentation analysis. Early, during the initial lag phase of the splicing reaction, the pre-mRNA sedimented heterogeneously but was detected in both 40S and 60S RNP complexes. An RNA substrate lacking a 3' splice site consensus sequence was not assembled into the 60S RNP complex. The two splicing intermediates, the first exon RNA species and an RNA species containing the intron and the second exon in a lariat configuration (IVS1-exon 2 RNA species), were found exclusively in a 60S RNP complex. These two splicing intermediates cosedimented under a variety of conditions, indicating that they are contained in the same RNP complex. The products of the splicing reaction, accurately spliced RNA and the excised IVS1 lariat RNA species, are released from the 60S RNP complex and detected in smaller RNP complexes. Sequence-specific RNA-factor interactions within these RNP complexes were evidenced by the preferential protection of the pre-mRNA branch point from RNase A digestion and protection of the 2'-5' phosphodiester bond of the lariat RNA species from enzymatic debranching. The various RNP complexes were further characterized and could be distinguished by immunoprecipitation with anti-Sm and anti-(U1)RNP antibodies.
...
PMID:Ribonucleoprotein complex formation during pre-mRNA splicing in vitro. 294 39


1 2 3 Next >>

---