Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro

The virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells was separated from the three host nuclear RNA polymerases by DEAE-Sephadex chromatography and then purified through two more steps heparin-agarose chromatography and glycerol...

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Bibliographic Details
Published in:Virology (New York, N.Y.) N.Y.), 1996-02, Vol.216 (1), p.12-19
Main Authors: Beniya, H. (Texas AandM University, College Station, TX.), Funk, C.J, Rohrmann, G.F, Weaver, R.F
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Animals
AUTOGRAPHA CALIFORNICA
Base Sequence
Cell Line
Centrifugation, Density Gradient
Chromatography, Agarose
Chromatography, Ion Exchange
CULTIVO DE CELULAS
CULTURE DE CELLULE
DNA, Viral
DNA-Directed RNA Polymerases - biosynthesis
DNA-Directed RNA Polymerases - isolation & purification
DNA-Directed RNA Polymerases - metabolism
Enzyme Induction
EXPERIMENTACION IN VITRO
EXPERIMENTATION IN VITRO
Gene Expression Regulation, Viral
Heparin
Lepidoptera
Molecular Sequence Data
Noctuidae
nuclear polyhedrosis virus
Nucleopolyhedrovirus - enzymology
Nucleopolyhedrovirus - genetics
PURIFICACION
PURIFICATION
Spodoptera - cytology
SPODOPTERA FRUGIPERDA
Transcription, Genetic
TRANSFERASAS
TRANSFERASE
VIRUS POLIEDROSIS NUCLEAR
VIRUS POLYEDROSE NUCLEAIRE
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Summary:The virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells was separated from the three host nuclear RNA polymerases by DEAE-Sephadex chromatography and then purified through two more steps heparin-agarose chromatography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayed in vitro for the ability to perform accurate initiation of transcription on a late (p6.9) and a very late (polyhedrin) template using primer extension analysis. In each case, the ability to accurately initiate transcription of these genes coincided with the virus-induced polymerase activity. Only after the glycerol gradient ultracentrifugation step did significant amounts of nonspecific late initiation occur, but specific late initiation was still readily detectable, suggesting that there is a limited number of late transcription factors, or that the factors are stably bound in a complex. After the glycerol gradient ultracentrifugation step, SDS-PAGE showed fewer than 10 prominent polypeptides remaining in the active fractions, which suggests a high degree of purity of the transcription machinery
ISSN:0042-6822
1096-0341
DOI:10.1006/viro.1996.0029