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Titolo:
SPECIALIZATION OF THE DNA-CLEAVING ACTIVITY OF A GROUP-I RIBOZYME THROUGH IN-VITRO EVOLUTION
Autore:
TSANG J; JOYCE GF;
Indirizzi:
SCRIPPS CLIN & RES INST,DEPT CHEM,10666 N TORREY PINES RD LA JOLLA CA92037 SCRIPPS CLIN & RES INST,DEPT CHEM LA JOLLA CA 92037 SCRIPPS CLIN & RES INST,DEPT MOL BIOL LA JOLLA CA 92037 SCRIPPS CLIN & RES INST,SKAGGS INST CHEM BIOL LA JOLLA CA 92037
Titolo Testata:
Journal of Molecular Biology
fascicolo: 1, volume: 262, anno: 1996,
pagine: 31 - 42
SICI:
0022-2836(1996)262:1<31:SOTDAO>2.0.ZU;2-L
Fonte:
ISI
Lingua:
ENG
Soggetto:
TETRAHYMENA RIBOZYME; RNA SUBSTRATE; BINDING-SITE; ENZYME; RIBOSE; POLYMERASE; SECONDARY; CLEAVAGE; INVITRO; INTRON;
Keywords:
CATALYTIC RNA; DNA CLEAVAGE; IN VITRO EVOLUTION; RIBOZYME;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
32
Recensione:
Indirizzi per estratti:
Citazione:
J. Tsang e G.F. Joyce, "SPECIALIZATION OF THE DNA-CLEAVING ACTIVITY OF A GROUP-I RIBOZYME THROUGH IN-VITRO EVOLUTION", Journal of Molecular Biology, 262(1), 1996, pp. 31-42

Abstract

In an earlier study, an in vitro evolution procedure was applied to alarge population of variants of the Tetrahymena group I ribozyme to obtain individuals with a 10(5)-fold improved ability to cleave a target single-stranded DNA substrate under simulate physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selectionfor DNA cleavage with a negative selection against RNA binding. After36 ''generations'' of in vitro evolution, the evolved population showed an similar to 100-fold increase in the ratio of DNA to RNA-cleavageactivity. Site-directed mutagenesis experiments confirmed the selective advantage of two covarying mutations within the catalytic core of the ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average of 28 mutations relative to the wild-type that are responsible for the altered phenotype. (C) 1996 Academic Press Limited

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Documento generato il 21/09/20 alle ore 03:35:39