MersV1, Main, Curation, bibRecord, 003732

Infectious Pancreatic Necrosis Virus RNA Cleavage In Vitro by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators

Identifieur interne : 003732 ( Main/Curation ); précédent : 003731; suivant : 003733

Infectious Pancreatic Necrosis Virus RNA Cleavage In Vitro by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators

Auteurs : Jyh-Yih Chen ; Jyh-Yeu Chen ; Wan-Ken Chu ; Huey-Nan Wu ; Ya-Li Hsu ; Jen-Leih Wu

Source :

Marine Biotechnology [ 1436-2228 ] ; 2000-07-01.

RBID : ISTEX:D669F5B9B7386DE022ECC32C294BCCE860FB9CC2

English descriptors

KwdEn :
- Key words: Infectious pancreatic necrosis virus (IPNV), ribozyme, oligonucleotide facilitator, RNA cleavage, catalysis..

Abstract

Abstract:: Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus, has a bisegmented double-stranded RNA genome consisting of a 3.2-kb A segment and a 2.9-kb B segment. To determine the function of IPNV's viral proteins and to study the effects of viral RNA cleavage by hammerhead ribozymes, we cloned and sequenced the IPNV E1S strain of the A segment. After sequencing, we continued to study the virus pathogens inhibited by ribozyme cleavage and analyzed the cleavage of the virus RNA in vitro. The templates (VP2, 1220 bp) for in vitro transcription of S569 and S969 (substrates 569 and 969 bp in length) were synthesized by polymerase chain reaction. The DNA templates of hammerhead ribozymes targeted different sites in the partial sense RNA of IPNV. These templates were chemically synthesized RNAs prepared by runoff transcription of amplification products or synthetic DNA templates containing a T7 RNA polymerase promoter, and were used to characterize several properties of the cleavage reaction at 25°C in 12 mM Mg2+. Under this condition (25°C, 12 mM Mg2+), the hammerhead ribozymes formed an estimated fraction of product during the reaction of only 30% in cleaving long RNA substrates in vitro. Short DNA facilitators (12 or 24-mers) that bind adjacent to either the 3′ or 5′ end of the ribozyme enhanced the rate of cleavage of the long RNA substrates containing 569 and 969 nucleotides, respectively, in trans. The hammerhead ribozymes with 3′-end facilitators reacted more efficiently (i.e., 65%).

Url:

https://api.istex.fr/ark:/67375/VQC-6H28BN0M-J/fulltext.pdf

DOI: 10.1007/s101269900033

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Jyh-Yih Chen

<affiliation><wicri:noCountry code="subField">TW</wicri:noCountry>
</affiliation>

Jyh-Yeu Chen

<affiliation><wicri:noCountry code="subField">TW</wicri:noCountry>
</affiliation>

Wan-Ken Chu

<affiliation><wicri:noCountry code="subField">TW</wicri:noCountry>
</affiliation>

Huey-Nan Wu

<affiliation><wicri:noCountry code="subField">TW</wicri:noCountry>
</affiliation>

Ya-Li Hsu

<affiliation><wicri:noCountry code="subField">TW</wicri:noCountry>
</affiliation>

Jen-Leih Wu

<affiliation><wicri:noCountry code="subField">TW</wicri:noCountry>
</affiliation>

Le document en format XML

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<front><div type="abstract" xml:lang="en">Abstract:: Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus, has a bisegmented double-stranded RNA genome consisting of a 3.2-kb A segment and a 2.9-kb B segment. To determine the function of IPNV's viral proteins and to study the effects of viral RNA cleavage by hammerhead ribozymes, we cloned and sequenced the IPNV E1S strain of the A segment. After sequencing, we continued to study the virus pathogens inhibited by ribozyme cleavage and analyzed the cleavage of the virus RNA in vitro. The templates (VP2, 1220 bp) for in vitro transcription of S569 and S969 (substrates 569 and 969 bp in length) were synthesized by polymerase chain reaction. The DNA templates of hammerhead ribozymes targeted different sites in the partial sense RNA of IPNV. These templates were chemically synthesized RNAs prepared by runoff transcription of amplification products or synthetic DNA templates containing a T7 RNA polymerase promoter, and were used to characterize several properties of the cleavage reaction at 25°C in 12 mM Mg2+. Under this condition (25°C, 12 mM Mg2+), the hammerhead ribozymes formed an estimated fraction of product during the reaction of only 30% in cleaving long RNA substrates in vitro. Short DNA facilitators (12 or 24-mers) that bind adjacent to either the 3′ or 5′ end of the ribozyme enhanced the rate of cleavage of the long RNA substrates containing 569 and 969 nucleotides, respectively, in trans. The hammerhead ribozymes with 3′-end facilitators reacted more efficiently (i.e., 65%).</div>
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Infectious Pancreatic Necrosis Virus RNA Cleavage In Vitro by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators

Infectious Pancreatic Necrosis Virus RNA Cleavage In Vitro by Hammerhead Ribozymes and Enhancement of Ribozyme Catalysis by Oligonucleotide Facilitators

Source :

English descriptors

Abstract

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