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

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Query: EC:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.
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PMID:Rabbit globin mRNA: analysis of T1 RNAse digestion fragments. 6 35

In a patient with homozygous betaO-thalassemia in whom studies of reticulocyte hemoglobin synthesis showed no beta-globin chain synthesis in vivo and in vitro, molecular hybridization studies revealed RNA sequences complementary to beta-globin cDNA. The fact that these sequences were authentic beta-globin mRNA was shown by fingerprint analysis of T1 ribonuclease-digested mRNA and by sequencing of oligonucleotides unique to beta-globin mRNA. The beta-mRNA that failed to direct beta-globin chain synthesis was not detectably shortened or degraded and contained poly(A) sequences.
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PMID:Authentic beta-globin mRNA sequences in homozygous betaO-thalassemia. 26 54

A method to enrich large size DNA fragments obtained by digestion with rare cutting restriction endonucleases was developed and applied for the isolation of a 150 kb SfiI fragment containing the beta-globin gene cluster. The digested DNA is rendered single stranded at the ends by diffusing a strand specific exonuclease into an agarose plug containing DNA. The plug is melted and solution hybridization is then performed with a bridge RNA containing specific sequences from the end of a desired fragment linked to a common probe sequence. The common probe sequence is annealed to a biotinylated RNA and the resulting tripartite hybrid is retained onto a solid matrix containing avidin and specifically released by ribonuclease action. Enrichments of greater than 350 fold have been achieved consistently. Such directed purification of large DNA fragments without cloning can considerably expedite mapping and gene localization in a complex genome and facilitate the construction of sublibraries from defined regions of the genome.
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PMID:Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping. 233 56

The applicability of ribonuclease cleavage at mismatches in RNA:DNA duplexes (RNase cleavage method) for determining nucleotide variant rates has been examined in a Japanese population. DNA segments of various lengths obtained from 4 different regions of a normal and 3 thalassemic cloned human beta-globin genes were inserted into transcription vectors. Sense and antisense RNA probes uniformly labeled with 32P were prepared. When RNA probes of 771 nucleotides (nt) or less were hybridized with cloned DNAs and the resulting duplexes were treated with a mixture of RNases A and T1, the length of products agreed with theoretical values. Twelve possible mismatches were examined. Since both sense and antisense probes were used, uncleavable mismatches such as G:T and G:G which were made from one combination of RNA and DNA strands could be converted to the cleavable C:A and C:C mismatches, respectively, by using the opposite combination. Deletions and insertions of 1 (G), 4 (TTCT), 5 (ATTTT) and 10 (ATTTTATTTT) nt were easily detected. A polymorphic substitution of T to C at position 666 of the second intervening sequence (IVS2-666) of the beta-globin gene was detected using genomic DNAs from cell lines established from the peripheral B lymphocytes of 59 unrelated Japanese from Hiroshima or those amplified by polymerase chain reaction (PCR). The frequency of the gene with C at the IVS2-666 (allele C) was 0.48 and that of the gene with T (allele T) was 0.52. The associations of the 2 alleles were in agreement with Hardy-Weinberg proportions. No contradiction to Mendelian inheritance was observed in the results obtained from 11 family studies. Two new polymorphic substitutions of C to A and A to T were detected at nucleotide positions 1789 and 1945 from the capping site, respectively, using genomic DNAs amplified by PCR. The feasibility of the RNase cleavage method combined with PCR for large-scale screening of variation in chromosomal DNA is discussed.
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PMID:Detection of deletions, insertions and single nucleotide substitutions in cloned beta-globin genes and new polymorphic nucleotide substitutions in beta-globin genes in a Japanese population using ribonuclease cleavage at mismatches in RNA:DNA duplexes. 238 39

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.
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PMID:Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate. 244 24

We have previously described an English family with gamma delta beta-thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5' to the cap site to 350 basepairs 3' to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.
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PMID:The inactive beta globin gene on a gamma delta beta thalassemia chromosome has a normal structure and functions normally in vitro. 334 45

Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis. Analysis of every type of mismatch in many different sequence contexts indicates that more than 50 percent of all single base substitutions can be detected. The feasibility of this method for localizing base substitutions directly in genomic DNA samples is demonstrated by the detection of single base mutations in DNA obtained from individuals with beta-thalassemia, a genetic disorder in beta-globin gene expression.
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PMID:Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. 407 Oct 43

Poly(U) binds to globin mRNA in 0.1m-NaCl. Studies with ribonuclease digestion of this complex suggest that there are polyadenylate-rich sequences in the mRNA containing about 30-40 adenylate residues. The sequences appear to be homogenous and of approximately the same length for both alpha- and beta-globin mRNA. They are most likely located at the 3' terminus of the molecule.
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PMID:Interaction between polyuridylic acid and rabbit globin messenger ribonucleic acid. 435 10

The ability of purified U1 small nuclear RNA-protein complexes (U1 snRNPs) to bind in vitro to two RNAs transcribed from recombinant DNA clones by bacteriophage T7 RNA polymerase has been studied. A transcript which contains sequences corresponding to the small intron and flanking exons of the major mouse beta-globin gene is bound in marked preference to an RNA devoid of splice site sequences. The site of U1 snRNP binding to the globin RNA has been defined by T1 ribonuclease digestion of the RNA-U1 snRNP complex. A 15-17-nucleotide region, including the 5' splice site, remains undigested and complexed with the snRNP such that it can be co-precipitated by antibodies directed against the U1 snRNP. Partial proteinase K digestion of the U1 snRNP abolishes interaction with the globin RNA, indicating that the snRNP proteins contribute significantly to RNA binding. No RNA cleavage, splicing, or recognition of the 3' splice site by U1 snRNPs has been detected. Our results are discussed in terms of the probable role of U1 snRNPs in the messenger RNA splicing of eucaryotic cell nuclei.
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PMID:The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. 619 May 73

Interleukin 2 (IL2) mRNA has a short half-life in the cytoplasm of T lymphocytes, relative to most mRNA. We have discovered a candidate ribonuclease to account for the rapid turnover of IL2 mRNA in the cytosol of the human T lymphocyte cell line Jurkat. In partially purified form, this RNase is about 7 times as active on IL2 as on beta-globin mRNA. Pancreatic RNase, by contrast, does not show a significant preference for IL2 mRNA. Neither 5' capping, nor polyadenylation of the substrate mRNAs affects their degradation by the IL2-selective mRNase, whose activity is optimal in 0.5 mM Mg++ and 100 mM potassium acetate. The mRNase behaves like a protein of molecular weight 60-70,000 on gel chromatography, and is unusual in that it is insensitive to placental RNase inhibitor (RNasin). The mRNase cleaves IL2 mRNA at a small number of sites in the coding region, and IL2 mRNA containing only the coding region and 36 nucleotides of the 3'-noncoding region competes efficiently with full-length IL2 mRNA for the mRNase, whereas beta-globin mRNA does not.
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PMID:An RNasin-resistant ribonuclease selective for interleukin 2 mRNA. 844 10


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