Addressed fragmentation of RNA molecules

Stepanova, O.; Metelev, Valeri; Chichkova, Nina V.; Smirnov, V. D.; Родионова, Н. В.; Atabekov, J.G.; Bogdanov, Alexei A.; Shabarova, Z. A.

doi:10.1016/0014-5793(79)81280-6

Cited by 22 publications

(6 citation statements)

References 11 publications

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“…B.) in collaboration with Metelev, Stepanova, and others [3][4][5][6] to cut RNA in the short oligodeoxyribonucleotide binding site, i.e., in the short DNA-RNA hybrid.…”

Section: Introductionmentioning

confidence: 99%

An enzymatic approach for localization of oligodeoxyribonucleotide binding sites on RNA

Mankin¹,

Skripkin²,

Chichkova³

et al. 1981

FEBS Letters

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“…B.) in collaboration with Metelev, Stepanova, and others [3][4][5][6] to cut RNA in the short oligodeoxyribonucleotide binding site, i.e., in the short DNA-RNA hybrid.…”

Section: Introductionmentioning

confidence: 99%

An enzymatic approach for localization of oligodeoxyribonucleotide binding sites on RNA

Mankin¹,

Skripkin²,

Chichkova³

et al. 1981

FEBS Letters

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“…Furthermore, very large RNA molecules (such as messengers) can be probed by modifying them first and then cutting them into smaller pieces (28,29) for end labeling, strand scission, and gel analysis.…”

mentioning

confidence: 99%

Chemical probes for higher-order structure in RNA.

Peattie¹,

Gilbert²

1980

Proc. Natl. Acad. Sci. U.S.A.

400

265

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Three chemical reactions can probe the secondary and tertiary interactions of RNA molecules in solution. Dimethyl sulfate monitors the N-7 of guanosines and senses tertiary interactions there, diethyl pyrocarbonate detects stacking of adenosines, and an alternate dimethyl sulfate reaction examines the N-3 of cytidines and thus probes base pairing. The reactions work between 0C and 90'C and at pH 4.5-8.5 in a variety of buffers. As an example we follow the progressive denaturation of yeast tRNA"h terminally labeled with 32P as the tertiary and secondary structures sequentially melt out. A single autoradiograph of a terminally labeled molecule locates regions of higher-order structure and identifies the bases involved.The three-dimensional configuration of an RNA molecule determines many of its biological properties. Chemical reagents provide sensitive probes of the conformation of nucleic acids in solution, detecting such properties as base pairing, base stacking, or the shielding of reactive groups by tertiary structure or complex formation with a protein or ion. With a base-specific reagent and a terminally labeled polynucleotide, a single experiment can probe such properties of that base wherever it occurs in the molecule. Our base-specific reagents weaken the glycosyl bond between the base and the ribosyl moiety of the polynucleotide. Thus, an initial, limited, base-specific modification appears ultimately as a chemically induced strand scission. The distance of the strand scission from the terminal label locates the position of the attacked base'along the molecule. The resultant nucleotide fragments are electrophoretically separated by size on polyacrylamide gels and their lengths are determined by autoradiography. This approach underlies the chemical determination of the sequence of DNA (1) and RNA (2) molecules. It has also been used to study .Dimethyl sulfate alkylates the N-7 position of guanosines (6) and the N-3 position of cytidines (7); diethyl pyrocarbonate carbethoxylates the N-7 of adenosines (8). However, these reactions occur only if the sites are not involved in structural interactions and, hence, are available for chemical modification. Furthermore, the diethyl pyrocarbonate reaction appears to be sensitive to the stacking of adenosines. Thus, these reagents effectively probe conformation by providing structure-specific data as well as base-specific information. If an RNA molecule is modified under totally denaturing conditions, a full sequence spectrum is generated on the autoradiograph that displays the electrophoretically separated labeled fragments (2). However, if the molecule is probed under native or semidenaturing conditions, only a partial sequence spectrum.appears (see Fig. 1 for example). Thus, when the probing reactions are run in parallel with the chemical sequencing reactions, a single autoradiograph locates regions of higher-order structure and simultaneously identifies the bases involved. The RNA can be labeled before or after the initial reactions.By using yeast tRNA...

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“…The method of site-specific cleavage with RNase H has been applied to different positions in diverse high Mr RNAs (Stepanova et al, 1979;Donis-Keller, 1979;Metelev et al, 1980;Dolja et al, 1982;Rodionova et al, 1983;Atabekov et al, 1988). In the case of BSMV RNAs and BMV RNA 3 the cleavage was performed at internal poly(A) tracts at different positions in the two viral RNAs.…”

Section: Discussionmentioning

confidence: 99%

Deletion of the Intercistronic Poly(A) Tract from Brome Mosaic Virus RNA 3 by Ribonuclease H and Its Restoration in Progeny of the Religated RNA 3

Карпова

Tyulkina

Atabekov

et al. 1989

Journal of General Virology

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SUMMARYThe dicistronic genomic RNA 3 of brome mosaic virus (BMV) was used in experiments on site-specific cleavage by RNase H and subsequent religation of large BMV RNA 3 fragments with T4 RNA ligase. BMV RNA 3 was cleaved at the intercistronic poly(A) tract into two fragments: a long (L-BMV 3) Y-terminal fragment (Mr 0.40 x 106) containing the 3a gene, and a short (Sh-BMV 3) fragment (Mr 0.28 x 106) containing the coat protein gene and the Y-terminal tRNA-like tyrosineaccepting structure. Two or three adenylate residues were present at the 5' end of Sh-BMV 3 and one adenylate at the Y end of L-BMV 3. After religation of these RNA fragments BMV RNA 3 was constructed with a deletion including the entire intercistronic poly(A) tract but not the flanking sequences. The religated RNA 3 replicated in wheat plants co-inoculated with BMV RNA 1 and RNA 2. The normal poly(A) tract was restored in progeny BMV RNA 3 during the course of replication.

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Addressed fragmentation of RNA molecules

Cited by 22 publications

References 11 publications

An enzymatic approach for localization of oligodeoxyribonucleotide binding sites on RNA

An enzymatic approach for localization of oligodeoxyribonucleotide binding sites on RNA

Chemical probes for higher-order structure in RNA.

Deletion of the Intercistronic Poly(A) Tract from Brome Mosaic Virus RNA 3 by Ribonuclease H and Its Restoration in Progeny of the Religated RNA 3

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