RNase digestion

Q-Sepharose was the separation medium chosen for preparative purification of plasmid pUC18.41 Cell proteins, fragments from RNase digestion of RNA, open coil plasmid and a denatured form of the plasmid were... 

Create an RNAse working buffer. RNAse T1 and/or RNAse A can be used at various concentrations. In Fig. 6.3A, RNAse T1 was diluted 1 10 in RNAse digestion buffer. 

Comments There are several suggested controls for this assay, including use of yeast total RNA as a negative control (check for probe species specificity) and a no RNAse control to determine probe stability. In Fig. 6.3A, the positive control marker lane was produced by addition of R-luc-4 sites or F-luc mRNA only to the assay. Also, optimal times for RNAse digestion will vary from probe to probe. In addition, for maximum sensitivity a probe with high specific activity is preferable (yet still in molar excess to the mRNA). 

RNase protection is used to detect the RNase-sensitive site in a synthetic RNA probe that has been hybridized with the DNA fragment to be examined. If a particular mutation lies within the DNA fragment, then the RNA probe is mismatched at the site of the mutation where RNase digestion occurs. 

Ribonucleases are macromolecules and therefore sensitive to steric hindrance especially from the proteins in the RNA-protein complexes and from the intramolecular structures. The analysis performed with the ribonucleases described in this section provides valuable information on the gross folding of an RNA, and in many cases the mild conditions for RNase digestions are optimal for RNA-protein complexes. 

ON THE LOCALIZATION OF RNA IN THE PARTICLE. The availability of particulate RNA to RNase digestion may be a result of the localization of RNA on the surface of a particle. This possibility was studied further in experiments on the interaction of particles with free RNA, and it was noted that a small additional amount of free RNA may combine with preexisting 308 particles. This reaction is specific for... 

The kinetics of RNase digestion have been studied with particles containing the intrinsic D-RNA labeled with and exogeneous D-RNA, combined with the particles, labeled with The kinetics of digestion of the RNA with RNA were found to be exactly the same (Lukanidin, 1969). This means that the intrinsic RNA of the 308 particles and the RNA artificially combined with the particles are similarly localized, probably on the surface of the particle. Although this conclusion is somewhat tentative, it provides a good explanation for several physical-chemical properties of the particles. 

RNase digestion products of 5S rRNA of S. acidocaldarius (b) Post-source decay mass spectrum obtained from the precursor ion... 

Durairaj, A. and Limbach, P.A. (2008) Matrix-assisted laser desorption/ ionization mass spectrometry screening for pseudouridlne in mixtures of small RNAs by chemical derivatization, RNase digestion and signature products. Rapid Commun. Mass Spectrom., 22 (23), 3727-3734. 

Figure 1 shows the proteins contained in various subnuclear fractions. Histones were not detectable in the isolated nuclear matrix by Coomassie blue staining. But these known acceptor proteins for ADP-ribose were found in the ammonium sulfate extract. Autoradiography revealed that great amounts of the self-modified poly(ADP-ribose) synthetase - in accordance with the results of Table 1 - were released by DNase, RNase digestion of the isolated nuclei (not shown). 

The number of all oligoribonucleotides generated by complete RNase digestion. 

In order to analyze the irradiated complexes for reaction products among proteins and ribosomal RNA, proteins are extracted with acetic acid following the method of Hardy et al. From a reaction mixture irradiated at 13° for 3 hr and digested with RNase as described above, 200-j l aliquots are shaken at 4 for 40 min after addition of 400 /d of acetic acid and 20 /J of 1 Af MgClj. After centrifugation (10 min, 20,000 g), the supernatant liquid is plated on GF/A filters. Macromolecular material is precipitated with 10% trichloroacetic acid at 4°. The rRNA pellet is dissolved in 100 / 1 of 150 roM NaCl, 50 mJf EDTA pH 7.0, 0.5% SDS, and precipitated on GF/A filters with 10% trichloroacetic acid as described above. The results (Table II) show that iV-acyl-Phe-tRNA is covalently bound to rRNA rather than to ribosomal proteins. When RNase digestion is carried out as described above but with a 10-fold increased quantity of RNase A (= 1 ftg/100 fil), no radioactivity is found in the rRNA fraction, indicating hydrolysis of the reaction product. 

Prewarm the washing solution and the RNase digestion buffer to 55"C and 37°C, respectively. 

Up to 8% of the newly synthesized RNA present in single-stranded form anneals to poUovirus plus strand and up to 9% of the added poliovirus plus-strand RNA is protected from RNase digestion by the newly synthesized RNA. The conversion of both viral RNA and newly synthesized RNA from an RNase-sensitive to an RNase-resistant RNA is dependent on the relative proportions of the RNA. 

Another approach to the determination of sequence heterogeneity of the bound 80 S and free cytoplasmic rRNA involved the fractionation of pancreatic RNase digests of rRNA by two-dimensional column chromatography. In a double-label experiment, free 26 S rRNA labeled with [ C]uracil and bound 26 S rRNA labeled with PH]uracil were mixed, digested with pancreatic RNase, and the oligonucleotide mixture was separated according to chain length on a column of DEAE-cellulose in... 

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