RNase Digestion Buffer

2013 ◽  
Vol 2013 (3) ◽  
pp. pdb.rec074146
Keyword(s):  
Rnase Digestion

scholarly journals Rabbit globin mRNA: analysis of T1 RNAse digestion fragments.

1977 ◽  
Vol 252 (10) ◽  
pp. 3446-3458
Author(s):  
G V Paddock ◽  
R Poon ◽  
H C Heindell ◽  
J Isaacson ◽  
W Salser
Keyword(s):  
Globin Mrna ◽  
Rnase Digestion ◽  
Mrna Analysis

Induction of implantation in the rat by intraparametrial injection of uterine RNA from oestrogen-treated animals

1982 ◽  
Vol 93 (3) ◽  
pp. 397-NP ◽  
Author(s):  
B. Lejeune ◽  
F. Puissant ◽  
M. Camus ◽  
F. Leroy
Keyword(s):  
Messenger Rna ◽  
Rna Synthesis ◽  
Ovariectomized Rats ◽  
Pregnant Rats ◽  
Rnase Digestion

Crude RNA preparations from uteri of oestradiol-treated rats induced the implantation of delayed blastocysts when injected into the parametrium of ovariectomized pregnant rats. Treatment of donor animals with labelled oestradiol showed that this effect could not be due to contamination of the RNA extracts by oestradiol. RNase digestion of these extracts suppressed their capacity to induce implantation. Purified poly (A)-rich RNA from oestrogen-treated uteri failed to elicit implantation although it was capable of increasing epithelial height when repeatedly injected into uterine horns of ovariectomized rats. These results suggest that uterine RNA synthesis might somehow mediate the effects of oestrogen in causing implantation and that RNAs other than messenger RNA might be involved.

The Actions of Enzymes on Lampbrush Chromosomes

1962 ◽  
Vol s3-103 (62) ◽  
pp. 173-203
Author(s):  
H. C. MACGREGOR ◽  
H. G. CALLAN
Keyword(s):  
Proteolytic Enzymes ◽  
Lampbrush Chromosomes ◽  
Triturus Cristatus ◽  
Trichloracetic Acid ◽  
Protease Digestion ◽  
Lateral Loop ◽  
Peptic Digestion ◽  
Rnase Digestion ◽  
Break Chromosome ◽  
Loop Matrix

The chromomeres of lampbrush chromosomes of Triturus cristatus are Feulgen-positive; they therefore contain DNA. After removal of their DNA in boiling trichloracetic acid, the chromomeres stain with fast green at alkaline pH; they therefore contain basic protein. The lateral loops are Feulgen-negative; they stain with toluidine blue at acid pH, but much less intensely following RNase digestion; they therefore contain RNA. The spheres of chromosomes V and VIII do not contain RNA. Unfixed lampbrush chromosomes retain a life-like appearance in 0.07 M K/NaCl at pH 6.2; in this medium the nuclear sap disperses. As pH is raised to 8.5 the matrices of lateral loops dissolve but chromosome axes remain unbroken. Above pH 8.5 lampbrush chromosomes dissolve. As pH is lowered from 6.2, at between 5.8 and 5.4 coagulation occurs. If pH is rapidly reduced still further, a persistent relaxed condition sets in between 2.5 and 2. In concentrations of K/NaCl above 0.5 M lampbrush chromosomes dissolve. Lateral loop matrices dissolve in 0.25 M K/NaCl but chromosome axes remain unbroken. In concentrations of K/NaCl below 0.05 M lateral loop matrices dissolve, but even in distilled water chromosome axes remain unbroken. Trypsin at pH 6.2 and at pH 7.8 strips the matrices from lateral loops and occasionally breaks matrix fusions. It causes chromomeres to swell and coalesce, but fails to break chromosome axes. The action of ‘pan-protease’ resembles that of trypsin in all respects. Pepsin at pH 6.2 strips the matrices from lateral loops, but does not destroy chromomeres. At low pH peptic digestion is slow: the enzyme is attacking coagulated chromosomes; but if peptic digestion precedes a lowering of pH the overall outcome is a rapid solution of loop matrix, and under these conditions matrix and sphere fusions are broken. If trypsin or ‘pan-protease’ digestion precedes a lowering of pH there is a similarly rapid solution of loop matrix; thus the action is not specifically referable to pepsin. Under no conditions does pepsin break the axes of lampbrush chromosomes. RNase at pH 6.2 strips the matrices from lateral loops; this action is detectable at extreme dilution. RNase does not destroy chromomeres, nor does it break chromosome axes. If tryptic digestion follows RNase digestion this too fails to break chromosome axes. Unlike the proteolytic enzymes and RNase, DNase at pH 6.2 breaks the fibril between adjacent chromomeres, and it also breaks the axes of lateral loops. Contrary to Mazia's experience with salivary gland chromosomes, versene does not break the axes of lampbrush chromosomes even when applied in media of low electrolyte concentration. These results indicate that uninterrupted fibres of DNA run throughout the lengths of lampbrush chromosomes.

scholarly journals A unique ribonucleoprotein complex assembles preferentially on ecdysone-responsive sites in Drosophila melanogaster.

1993 ◽  
Vol 13 (9) ◽  
pp. 5323-5330 ◽  
Author(s):  
S A Amero ◽  
M J Matunis ◽  
E L Matunis ◽  
J W Hockensmith ◽  
G Raychaudhuri ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Cell Extract ◽  
Sequence Motifs ◽  
Drosophila Cell ◽  
Rnase Digestion

The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.

Isolation and translation in vitro of poly(A)+ RNA from the free-living nematode Panagrellus silusiae

1979 ◽  
Vol 57 (4) ◽  
pp. 330-335 ◽  
Author(s):  
J. Scott Noble ◽  
J. Pasternak
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mean Value ◽  
Amino Acid Incorporation ◽  
Free Living ◽  
Free Living Nematode ◽  
Rnase Digestion ◽  
Fold Stimulation ◽  
Cellulose Column

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)− RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)− RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate – polyacrylamide gels. Poly(A)− RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.

scholarly journals A Set of Novel RNAs Transcribed from the Chloroplast Genome Accumulates in Date Palm Leaflets Affected by Brittle Leaf Disease

2008 ◽  
Vol 98 (3) ◽  
pp. 337-344 ◽  
Author(s):  
J. Marqués ◽  
Z. G. N. Fadda ◽  
N. Duran-Vila ◽  
R. Flores ◽  
J. M. Bové ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Date Palm ◽  
Diagnostic Value ◽  
Northern Hybridization ◽  
Date Palms ◽  
Leaf Disease ◽  
Southern Tunisia ◽  
Rnase Digestion

Brittle leaf disease or maladie des feuilles cassantes (MFC) is a lethal disorder of date palms that has assumed epidemic proportions in the oases of southern Tunisia. After a prolonged period during which palms are declining, the disease ends with the death of the palms. Whereas no pathogen could ever be associated with the disease, leaflets of affected palms have been previously shown to be deficient in manganese. Analysis of RNA preparations from leaflets of MFC-affected palms revealed the presence of a set of novel RNAs (MFC-RNAs) of sense and antisense polarities, which are homologous to various regions of the date palm chloroplast genome, such as the regions containing genes rrn5S-trnR(ACG) and trnM(CAU)-atpE. In the RNA preparations obtained from leaflets of affected palms, some of these RNAs are present as double-stranded species (MFC-dsRNAs), as witnessed by results from cellulose chromatography, end labeling, RNase digestion, and northern hybridization with strand specific probes. These MFC-RNAs represent a novel type of host-derived RNAs, and their presence in MFC-affected date palms is of diagnostic value.

scholarly journals Nuclear Matrix-like Filaments and Fibrogranular Complexes Form through the Rearrangement of Specific Nuclear Ribonucleoproteins

2000 ◽  
Vol 11 (5) ◽  
pp. 1547-1554 ◽  
Author(s):  
Jia-huai Tan ◽  
John C. Wooley ◽  
Wallace M. LeStourgeon
Keyword(s):  
Nuclear Matrix ◽  
Disulfide Bridge ◽  
Filament Formation ◽  
Hnrnp Proteins ◽  
Nuclear Extracts ◽  
Residual Material ◽  
Scanning Transmission ◽  
Rnase Digestion ◽  
Nuclear Ribonucleoprotein ◽  
Mrna Binding

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)3B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 μm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the “nuclear matrix.” Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.

scholarly journals AbmR is a mycobacterial dual-function transcription factor and ribonucleoprotein with distinct DNA and RNA-binding determinants

2021 ◽  
Author(s):  
Roxie C. Girardin ◽  
Janice Pata ◽  
Xiaohong Qin ◽  
Haixin Sui ◽  
Kathleen A. McDonough
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Rna Binding ◽  
Rna Stability ◽  
Dual Function ◽  
Dna And Rna ◽  
Binding Function ◽  
Rnase Digestion ◽  
Binding Determinants ◽  
Dna And Rna Binding

ABSTRACTThe bacterium Mycobacterium tuberculosis (Mtb) must adapt to myriad host-associated stressors. A recently identified transcription factor, AbmR (ATP-binding mcr11-regulator), regulates expression of an essential stress-responsive small RNA (Mcr11) and inhibits the growth of Mtb. Previously, AbmR was found to make 39S complexes of unknown function. Here we report that AbmR 39S complexes are comprised of AbmR and co-purifying RNAs and that RNA-binding inhibits AbmR’s DNA-binding function. While AbmR binds DNA and regulates gene expression in a sequence specific manner, RNA-binding is not sequence specific. Amino acid R146 is important for DNA-binding but completely dispensable for RNA-binding and 39S complex formation, establishing that the RNA- and DNA-binding functions of AbmR are distinct. RNA bound by AbmR was protected from RNase digestion, supporting an RNA modulatory function for the 39S complex. We also found that abmR is required for optimal survival during treatment with the ATP-depleting antibiotic bedaquiline, which is associated with extended RNA stability. These data establish a paradigm wherein a transcription factor assembles into large complexes to transition between mutually exclusive DNA-binding gene regulatory and RNA-binding RNA modulatory functions. Our findings indicate that AbmR is a dual-function protein that may have novel RNA regulatory roles in stress adapted Mtb.

scholarly journals An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture).

1995 ◽  
Vol 15 (6) ◽  
pp. 3363-3371 ◽  
Author(s):  
I Edery ◽  
L L Chu ◽  
N Sonenberg ◽  
J Pelletier
Keyword(s):  
Selection Procedure ◽  
Full Length ◽  
Initiation Factor ◽  
Cdna Libraries ◽  
Eukaryotic Initiation Factor 4E ◽  
Rna Duplexes ◽  
Rnase Digestion ◽  
Specific Retention ◽  
Support Matrix

The ability to generate cDNA libraries is one of the most fundamental procedures in contemporary molecular biology. One of the major drawbacks of current methods is that most cDNAs present in any given library are incomplete, rendering the characterization of genes an inefficient and time-consuming task. We have developed an affinity selection procedure using a fusion protein containing the murine cap-binding protein (eukaryotic initiation factor 4E), coupled to a solid support matrix, that allows for the purification of mRNAs via the 5' cap structure. When combined with a single-strand-specific RNase digestion step, specific retention of complete cDNA-RNA duplexes following first-strand synthesis is achieved. This method can be used to generate cDNA libraries in which polyadenylated and nonpolyadenylated mRNAs are equally represented and to enrich for full-length or 5'-end clones, thus facilitating cDNA cloning and promoter mapping.

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